Neutrophil dysfunction may arise from (1) the absence of antibodies or complement components required to opsonize microorganisms, an interaction that provides a chemotactic signal; (2) the abnormalities of cytoplasmic and granule movement that alter the chemotactic response or that result in abnormalities of the plasma membrane affecting the cells in terms of capability to modulate movement; and (3) defective microbicidal capability. Comprehensive reviews of these syndromes are available to the interested reader.266,267,268
ABNORMALITIES OF THE SIGNAL MECHANISM AS A RESULT OF ANTIBODY AND COMPLEMENT DEFECTS OR IMPAIRMENT OF PATTERN RECEPTOR RECOGNITION
Because the synergistic action of immunoglobulins and complement proteins creates the opsonins that coat microorganisms and stimulate the development of chemotactic factors, a deficiency of either one may result in impaired neutrophil function. The most profound disturbances arise from abnormalities in C3, because this protein is the focal point for generation of opsonins and chemotactic factors (Chap. 19).269,270,271 Opsonins such as C3b, generated from cleavage of C3, serve to coat bacteria. Opsonization in general refers to the coating of pathogens by serum proteins such that they are more likely to be ingested. Activation of C3 can occur in the absence of an antibody or the classical complement components C1, C2, and C4; thus, disorders of these latter molecules result in less-severe clinical conditions. C3 deficiency is inherited as an autosomal recessive disorder. Homozygotes have undetectable serum levels of C3 and suffer from recurrent severe pyrogenic infections, whereas asymptomatic heterozygotes have half the normal values.
A functional deficiency of C3 protease resulting in severe pyrogenic infections also is seen in patients with a deficiency in C3b inactivator, a protein inhibitor of the alternative complement pathway. Unchecked activation of this pathway leads to hypercatabolism of C3 and factor B.272 Properidin deficiency also results in a functional deficiency in C3.273 Properidin is a serum protein that belongs to the alternative complement pathway; it is involved in the stabilization of the enzyme complex C3bBb. The protein is a multimeric glycoprotein with a subunit Mr of 56,000, the gene of which has been cloned.274 Absence of properidin is associated with severe, often fatal, pyrogenic infections, often with meningococci.
Approximately 5 percent of the population have low serum levels of mannose-binding lectin (MBL),275 a serum lectin secreted by the liver that binds mannose sugars present and on the surface of bacteria, fungi, and some viruses. MBL is one of the soluble collectin effector proteins that contribute to the basic armamentarium of innate immunity. MBL can function as an opsonin when bound to the surfaces by activating the complement cascade. A deficiency of MBL has been reported in infants with frequent unexplained infection, chronic diarrhea, and otitis media.275 Other studies have identified an increased susceptibility to infection by specific pathogens in MBL-deficient individuals, including human immunodeficiency virus, Plasmodium falciparum, Cryptosporidium parvum, and Neisseria meningitidis.276 The deficiency in MBL largely results from three relatively common single-point mutations in exon 1 of the gene, which leads to the failure of MBL to activate complement.277 In addition, the protein also modulates disease severity, at least in part through complex, dose-dependent influences on cytokine production.
Phagocytes, including neutrophils, express a large number of cell surface proteins that play crucial functional roles in their biology. Microbial PRRs are an essential component of innate immunity, in which they recognize and detect PAMPs, resulting in activation of neutrophils and other phagocytes. The mammalian TLR family comprises an important class of PRRs, which recognize a wide range of microbial pathogens and pathogen-related products. At least 12 different TLRs that can be found on mononuclear phagocytes have been described.278 TLRs signal via MyD88, an adapter protein. MyD88 deficiency in humans can lead to recurrent infections with both Gram-positive and Gram-negative infections, thereby indicating a role for both mononuclear cells and neutrophils in host defense in the MyD88-deficient state.279
Because a large number of chemoattractants are generated during inflammation, it is difficult to establish the relative significance of a given individual component. Furthermore, chemotactive factors and opsonins are involved in the activity of both neutrophils and mononuclear phagocytes. Therefore, it is not clear whether the clinical consequences of disorders involving these substances are unique to one or the other of these phagocytic cells. Patients with antibody- or complement-deficient syndromes suffer mainly from infections with encapsulated pathogens such as Haemophilus influenzae, pneumococci, streptococci, and meningococci.280 Furthermore, splenectomized individuals deprived of an organ rich in mononuclear phagocytes have a small, but finite risk of sepsis because of the same microorganisms. Encapsulated pathogens characteristically are not associated with neutropenic states. Antibody coating of encapsulated organisms facilitates their ingestion by mononuclear phagocytes, but may be less important for their ingestion by neutrophils.
ABNORMALITIES OF THE CELLULAR RESPONSES AS THE RESULTS OF DEFECTS IN CYTOPLASMIC MOVEMENT
Definition and History This rare autosomal recessive disease was initially recognized as one in which neutrophils, monocytes, and lymphocytes contained giant cytoplasmic granules.281 Chédiak-Higashi syndrome (CHS) is now recognized as a disorder of generalized cellular dysfunction characterized by increased fusion of cytoplasmic granules.282 Pigmentary dilution affecting the hair, skin, and ocular fundi results from pathologic aggregation of melanosomes and is associated with failure of decussation of the optic and auditory nerves (Table 66–2).283 Patients with this syndrome exhibit an increased susceptibility to infection, which begins in infancy. Infections most commonly involve the skin and respiratory systems. The susceptibility to infection can be explained in part through defects in neutrophil chemotaxis, degranulation, and bactericidal activity.281 The presence of giant granules in the neutrophil interferes with their ability to traverse narrow passages between endothelial cells. Other features of the disease include neutropenia, thrombocytopathy,284 natural killer cell abnormalities,281,285 and peripheral neuropathies.286 Similar genetic syndromes have been described in mice, mink, cats, rats, cattle, and killer whales.286
Table 66–2.Clinical Disorders of Neutrophil Function ||Download (.pdf) Table 66–2. Clinical Disorders of Neutrophil Function
|Disorder ||Etiology ||Impaired Function ||Clinical Consequence |
|DEGRANULATION ABNORMALITIES |
|Chédiak-Higashi syndrome ||Autosomal recessive; disordered coalescence of lysosomal granules; responsible gene is CHSI/LYST which encodes a protein hypothesized to regulate granule fusion ||Decreased neutrophil chemotaxis; degranulation and bactericidal activity; platelet storage pool defect; impaired NK function, failure to disperse melanosomes ||Neutropenia; recurrent pyogenic infections, propensity to develop marked hepatosplenomegaly as a manifestation of the hemophagocytic syndrome |
|Specific granule deficiency ||Autosomal recessive; functional loss of myeloid transcription factor arising from a mutation or arising from reduced expression of Gfi-1 or C/EBPε, which regulates specific granule formation ||Impaired chemotaxis and bactericidal activity; bilobed nuclei in neutrophils; defensins, gelatinase, collagenase, vitamin B12-binding protein, and lactoferrin ||Recurrent deep-seated abscesses |
|ADHESION ABNORMALITIES |
|Leukocyte adhesion deficiency I ||Autosomal recessive; absence of CD11/CD18 surface adhesive glycoproteins (β2 integrins) on leukocyte membranes most commonly arising from failure to express CD18 mRNA ||Decreased binding of C3bi to neutrophils and impaired adhesion to ICAM-1 and ICAM-2 ||Neutrophilia; recurrent bacterial infection associated with a lack of pus formation |
|Leukocyte adhesion deficiency II ||Autosomal recessive; loss of fucosylation of ligands for selectins and other glycol conjugates arising from mutations of the GDP-fucose transporter ||Decreased adhesion to activated endothelium expressing ELAM ||Neutrophilia; recurrent bacterial infection without pus |
|Leukocyte adhesion deficiency III (LAD-1 variant syndrome) ||Autosomal recessive; impaired integrin function arising from mutations of FERMT3 which encodes kindlin-3 in hematopoietic cells; kindlin-3 binds to β-integrin and thereby transmits integrin activation ||Impaired neutrophil adhesion and platelet activation ||Recurrent infections, neutropenia, bleeding tendency |
|DISORDERS OF CELL MOTILITY |
|Enhanced motile responses; FMF ||Autosomal recessive gene responsible for FMF on chromosome 16, which encodes for a protein called “pyrin”; pyrin regulates caspase-1 and thereby IL-1β secretion; mutated pyrin may lead to heightened sensitivity to endotoxin, excessive IL-1β production, and impaired monocyte apoptosis ||Excessive accumulation of neutrophils at inflamed sites which may be the result of excessive IL-1β production ||Recurrent fever, peritonitis, pleuritis, arthritis, and amyloidosis |
|DEPRESSED MOTILE RESPONSES |
|Defects in the generation of chemotactic signals ||IgG deficiencies; C3 and properdin deficiency can arise from genetic or acquired abnormalities; mannose-binding protein deficiency predominantly in neonates ||Deficiency of serum chemotaxis and opsonic activities ||Recurrent pyogenic infections |
|Intrinsic defects of the neutrophil, e.g., leukocyte adhesion deficiency, Chédiak-Higashi syndrome, specific granule deficiency, neutrophil actin dysfunction, neonatal neutrophils; direct inhibition of neutrophil mobility, e.g., drugs ||In the neonatal neutrophil there is diminished ability to express β2 integrins and there is a qualitative impairment in β2-integrin function; ethanol, glucocorticoids, cyclic AMP ||Diminished chemotaxis; impaired locomotion and ingestion; impaired adherence ||Propensity to develop pyogenic infections; possible cause for frequent infections; neutrophilia seen with epinephrine arises from cyclic AMP release from endothelium |
|Immune complexes ||Bind to Fc receptors on neutrophils in patients with rheumatoid arthritis, systemic lupus erythematosus, and other inflammatory states ||Impaired chemotaxis ||Recurrent pyogenic infections |
|Hyperimmunoglobulin-E syndrome ||Autosomal dominant; responsible gene is STAT3 ||Impaired chemotaxis at times; impaired regulation of cytokine production ||Recurrent skin and sinopulmonary infections, eczema, mucocutaneous candidiasis, eosinophilia, retained primary teeth, minimal trauma fractures, scoliosis, and characteristic facies |
|Hyperimmunoglobulin-E syndrome ||Autosomal recessive; more than one gene likely contributes to its etiology ||High IgE levels, impaired lymphocyte activation to staphylococcal antigens ||Recurrent pneumonia without pneumatoceles sepsis, enzyme, boils, mucocutaneous candidiasis, neurologic symptoms, eosinophilia |
|MICROBICIDAL ACTIVITY |
|Chronic granulomatous disease ||X-linked and autosomal recessive; failure to express functional gp91phox in the phagocyte membrane in p22phox (autosomal recessive); other autosomal recessive forms of CGD arise from failure to express protein p47phox or p67phox ||Failure to activate neutrophil respiratory burst leading to failure to kill catalase-positive microbes ||Recurrent pyogenic infections with catalase-positive microorganisms |
|G6PD deficiency ||Less than 5% of normal activity of G6PD ||Failure to activate NADPH-dependent oxidase, and hemolytic anemia ||Infections with catalase-positive microorganisms |
|Myeloperoxidase deficiency ||Autosomal recessive; failure to process modified precursor protein arising from missense mutation ||H2O2-dependent antimicrobial activity not potentiated by myeloperoxidase ||None |
|Rac-2 deficiency ||Autosomal dominant; dominant negative inhibition by mutant protein of Rac-2–mediated functions ||Failure of membrane receptor–mediated O2 generation and chemotaxis ||Neutrophilia, recurrent bacterial infections |
|Deficiencies of glutathione reductase and glutathione synthetase ||Autosomal recessive; failure to detoxify H2O2 ||Excessive formation of H2O2 ||Minimal problems with recurrent pyogenic infections |
Although CHS carries the names of Moises Chédiak and Ototaka Higashi, the disorder was first described by Béguez César, a Cuban pediatrician in 1943. Initially characterized by neutropenia and abnormal granules in leukocytes, the syndrome was further delineated in 1948 by Steinbrinck’s description of a second case.287 In 1952, Chédiak reported the hematologic characteristics of the disorder,288 and in 1953 Higashi emphasized the giant peroxidase-containing granules within patients’ neutrophils.289 Besides the susceptibility to infections, patients often suffer a fatal lymphohistiocytic infiltration known as the accelerated phase occurring months from birth to several years later.290
Epidemiology By 2008, 300 cases worldwide had been described, with concentrations in the United States, Japan, northern Europe, and Latin America.286 Patients of African descent have also been described.
Etiology and Pathogenesis CHS is caused by a fundamental defect in granule morphogenesis that results in abnormally large granules in multiple tissues.282,291 Giant granules are seen in Schwann cells, leukocytes, and macrophages of the liver and spleen, and certain cells of the pancreas, gastric mucosa, kidney, adrenal gland, and pituitary gland.286 Giant melanosomes form and prevent the even distribution of melanin, which results in pigmentary dilution of the hair, skin, iris, and optic fundus. Although the giant lysosomes are the primary morphologic feature of the disorder, only cells relying on the secretion by these lysosomes manifest pathologic defects. In the early stages of myelopoiesis some of the normal-size azurophil granules coalesce to form giant granules that result in large secondary lysosomes that contain reduced content of hydrolytic enzymes, including proteinases, elastase, and cathepsin G.281 Many of the myeloid precursors die in the marrow, resulting in a moderate neutropenia, with white cell counts of about 2.5 × 109/L and absolute neutrophil counts ranging from 0.5 to 2.0 × 109/L.290 The marrow itself appears normal to hypercellular. In spite of the normal ingestion of particles and active oxygen metabolism, these neutrophils kill microorganisms relatively slowly. This delay reflects a slow and inconsistent delivery of diluted amounts of hydrolytic enzymes from the giant granules into the phagosomes, which may predispose the host to bacterial infection.291,292 In this syndrome, monocytes have the same functional derangements as neutrophils,281 and in an analogous fashion perforin-deficient natural killer (NK) cells show profoundly impaired cytotoxic activity and are unable to kill many targets.293
The CHS blood cell membranes are more fluid than cells of normal individuals,281,294 and the altered membrane structure could lead to defective regulation of membrane activation, as well as promoting fusion of neutrophil azurophilic granules with each other. Conceivably, changes in membrane fluidity may affect cell function by reducing expression of Mac-1 (CD11b/CD18). The altered membrane fluidity could result in elevated levels of intracellular cyclic adenosine monophosphate, which appears in this disorder and is reflected in the reduced chemotactic responses.281
The gene that is mutated in CHS is CHS1 (syn. LYST) found on chromosome 1q. Its size indicates a protein of more than 400 kDa is encoded.295 During early development, granule biogenesis is normal; with perforin in NK cells and granule enzymes in myeloid cells synthesized and routed correctly to the granules. However, once formed the granules fuse to form giant organelles.296 Several studies led to the suggestion that the enlarged lysosomes found in CHS cells are the results of abnormalities in membrane fusion, which could occur during the biogenesis of the lysosomes. It has been hypothesized that this CHS1 protein interacts with attachment proteins on lysosomes (v-SNAREs) and that this mutated protein leads to indiscriminate interactions with v-SNARE to yield uncontrolled fusion of lysosomes with each other.297
Clinical Features Characteristically patients with CHS have light skin and silvery hair. They frequently complain of solar sensitivity and photophobia. Other eye findings can include horizontal or rotatory nystagmus. Infections are common and involve the mucous membranes, skin, and respiratory tract. They are susceptible to both Gram-positive and Gram-negative bacteria, as well as fungi, with Staphylococcus aureus being the most common infecting organism.266 Attenuated NK function probably contributes to the increased susceptibility to infection as well. Neurologic signs and symptoms are variable in CHS and may include a peripheral and cranial neuropathy, autonomic dysfunction, weakness, and sensory deficit; and ataxia may also be a prominent feature.
Patients with CHS have prolonged bleeding times with normal platelet counts, resulting from impaired platelet aggregation associated with a deficiency of the storage pools of ADP and serotonin.284 Electron micrographs reveal normal numbers of α granules in platelets, but decreased numbers of platelet dense bodies.286
The accelerated phase of CHS is characterized by lymphocytic proliferation in the liver, spleen, marrow, and central nervous system. The accelerated phase may occur at any age and is now recognized as a genetic form of hemophagocytic lymphohistiocytosis (HLH).298 Typically, the patient develops hepatosplenomegaly and high fever in the absence of bacterial sepsis. The pancytopenia becomes worse at this stage, producing hemorrhage and an increased susceptibility to infection. The onset of the accelerated phase may be related to the inability of these patients to contain and control the Epstein-Barr virus (EBV) leading to HLH (Chap. 70). The lymphocyte expansion into the tissue is associated with excessive cytokine production and massive tissue necrosis and organ failure leading to the propensity to recurrent bacterial and viral infections, fever, and prostration usually resulting in death.298 At autopsy, the lymphohistiocytic infiltrates in the liver, spleen, and lymph nodes are extensive, but not neoplastic by histopathologic criteria.298
Laboratory Features The laboratory test diagnostic for CHS is examination of granular cell morphology. The pathognomonic feature is giant peroxidase-positive granules that can be seen in neutrophils.289 A microscopic examination of hair shafts reveal large, speckled pigment clumps as opposed to the normal pattern of finally divided pigment of melanin spread along the length of the shaft.286 Similar giant granules can occasionally be present in CML and acute myelocytic leukemia.286 Molecular diagnosis of CHS remains difficult and is not commercially available. Heterozygotes for CHS are considered completely normal and cannot be detected clinically or biochemically.
Differential Diagnosis The diagnosis of CHS should be considered in individuals with partial albinism, exaggerated bleeding, and recurrent infections. Patients with CHS must be distinguished from those patients with Griscelli syndrome (GS) and Hermansky-Pudlak syndrome (HPS).
GS is a rare disorder, arising from mutations in the RAB27A gene, and is defined by partial ocular and cutaneous albinism, variable cellular and humeral immunodeficiency, variable neurologic involvement, and the development of the accelerated phase. Individuals with GS lack giant granules in neutrophils and have large pigment clumps in hair shafts.286 HPS is a disorder of ocular and cutaneous albinism, bleeding diathesis arising from platelet dysfunction, and deposition of ceroid lipofuscin in various organs (Chap. 120). In contrast to CHS, HPS cells lack giant granules and the patients are not predisposed to recurrent infections.286
Therapy High-dose ascorbic acid (200 mg/day for infants, 2 g/day for adults) improves the clinical status of some patients with CHS in the stable phase.281 Although there is controversy regarding the efficacy of ascorbic acid, given the safety of the vitamin,286 it is reasonable to administer it to all patients. CHS presents a therapeutic dilemma, particularly when the accelerated phase begins. Prophylactic antibiotics do not prevent infections. The only potential for curative therapy for preventing the accelerated phase is marrow transplantation.299 Marrow transplantation reconstitutes normal hematopoietic and immunologic function and corrects the NK deficiency in patients before entering the accelerated phase.299 On the other hand, if the patient is actively in the accelerated phase, stem cell transplantation from a matched unrelated donor is associated with a poor prognosis.299 Ocular and cutaneous albinism are not corrected after transplantation, nor does transplantation prevent progressive neuropathies from occurring.300
Specific Granule Deficiency Specific granule deficiency (SGD) has been described in patients of both sexes and is inherited as an autosomal recessive disorder (see Table 66–2).281 Besides the absence of specific granules, the nuclei of the neutrophils are bilobed. Patients are afflicted with recurrent infections primarily involving the skin and lungs. S. aureus and Pseudomonas aeruginosa have been the most commonly observed pathogens, although Candida albicans also has been isolated. Specific granule-deficient neutrophils lack gelatinolytic activity in the tertiary granules; vitamin B12-binding protein, lactoferrin, hCAP-18, and collagenase in the specific granules; and defensins in the primary granules.301,302,303 This disorder also extends to eosinophils that lack the characteristic eosinophil granule proteins: major basic protein, eosinophilic cationic protein, and eosinophil-derived neurotoxin (Chap. 62).304 Thus, the disorder is a global defect in phagocytic granules rather than limited to specific granules, as suggested by its name. Neutrophils from these patients are defective in chemotaxis, possibly related to the absence of the intracellular pool of leukocyte adhesion molecules that normally reside in the tertiary and specific granules, and exhibit a mild defect in bactericidal activity, possibly related to the deficiency of the granule constituents, lactoferrin and defensins.301,305 The impairment in granule protein synthesis affecting the granulocytic cells appears secondary to the functional loss of the myeloid transcription factor, C/EBPε, which was identified in two patients.109,306 In another case of SGD, the expression of the transcription factor growth factor independence-1 (Gfi-1) was markedly reduced along with a heterozygous mutation of C/EBPε gene.307 It was suggested that the combined abnormalities blocked specific granule expression leading to the expression of the SGD phenotype. The defect is restricted to blood cells, as normal lactoferrin secretion has been demonstrated in the nasal secretions of an SGD patient despite the abnormality demonstrated in his neutrophils.302 The diagnosis of SGD is suggested by the presence of neutrophils devoid of specific granules but containing azurophilic granules on the blood film.281 The diagnosis can be confirmed by demonstrating a severe deficiency in either lactoferrin or hCAP-18. An acquired form of SGD can be observed in thermally injured patients or in individuals with myelodysplasia.281,308 Treatment of SGD is symptomatic, with the administration of parenteral antibiotics for acute infections and surgical drainage of refractory infections. With aggressive medical management, patients may survive into their adult years.
Leukocyte Adhesion Deficiency
Definition and History Leukocyte adhesion deficiency type I (LAD-1) is a rare autosomal recessive disorder of leukocyte function (see Table 66–2). More than 100 cases have been reported worldwide. The disease is characterized clinically by recurrent soft-tissue infections, delayed wound healing, and severely impaired pus formation despite striking blood neutrophilia.309 Individuals with this disorder have decreased or absent expression of a family of structurally and functionally related leukocyte surface glycoproteins designated CD11/CD18 complex (also referred to as the β2-integrin family of leukocyte adhesive proteins; Table 66–3). These proteins include LFA-1 (CD11a/CD18), Mo-1 or Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and p160,95 (CD11d/CD18).309 The CD11 subunits are integral membrane glycoproteins, each spanning the plasma membrane only once. They are approximately 40 percent homologous, suggesting that they arise from a common primordial gene.309 The three distinct genes encoding the α subunits occur in a cluster on chromosome 16, whereas the gene for the β subunit is located on chromosome 21.310
Table 66–3.Biologic and Clinical Features of Leukocyte Adherence Deficiencies 1 and 2 ||Download (.pdf) Table 66–3. Biologic and Clinical Features of Leukocyte Adherence Deficiencies 1 and 2
| ||Genetic Defect ||Leukocyte Functional Abnormalities ||Clinical Features ||Diagnosis |
|LAD-1 ||Molecular mutations affecting expression of the β2-integrin CD18 ||Neutrophils; adherence spreading, homotypic aggregation, chemotaxis receptor CR3 activities: C3bi binding affecting phagocytosis, respiratory burst, and degranulation in response to C3bi-coated particles* ||Autosomal recessive; delayed umbilical cord separation; neutrophilia; defective neutrophil migration into tissue; recurrent bacterial infections; impaired wound healing ||Flow cytometry for expression of CD11b/CD18 (Mac-1) |
| || ||Monocytes; adherence, CR3 activities || || |
| || ||Lymphocytes; cytotoxic || || |
| || ||T-lymphocyte activities; NK cytotoxic activities; blastogenesis || || |
|LAD-2 (CDG-IIc) ||Mutations affecting function of GDP-fucose transporter 1 resulting in defective glycosylation expression at the α1,3-position of selectin ligands including sLex and other fucosylated proteins requiring fucosylation ||Neutrophils; rolling mediated by sLex to endothelium; neutrophilia† ||Autosomal recessive; recurrent bacterial infections, periodontitis; growth retardation; developmental retardation; Bombay red cell phenotype ||Flow cytometry for leukocyte sLex (CD15) |
The initial clinical description in 1979 described six children and two families with findings of delayed separation of the umbilical cord and delayed healing at the site of detachment of the cord, recurrent infections despite neutrophilia, neutrophilia persisting during infection-free periods, and impaired neutrophil chemotaxis.311 The molecular basis for LAD-1 was first suggested when neutrophils from a patient with the disorder was found to lack a high-molecular-weight membrane glycoprotein.312 This finding suggested that the lack of the membrane protein impaired the neutrophil’s functional responses. In 1982, another patient was evaluated and it was confirmed that the membrane glycoprotein with a Mr of 150 kDa was missing.313 The normal parents and siblings of the proband exhibited intermediate quantities of the glycoprotein, which suggested the existence of a heterozygous carrier state. The disorder then became known as LAD. Subsequently, in 1984, a glycoprotein 150 was identified as one subunit of a glycoprotein that had two subunits that served as a receptor for a plasma complement component.314 This was followed by other investigations that found that two other related leukocyte membrane glycoproteins also were deficient. Each of the three glycoproteins was then determined to be heterodimers with one common subunit and one subunit unique to each glycoprotein.315 Synthesis of a defective subunit common to the three glycoproteins of CD11/CD18 complex resulted in loss of expression of all heterodimers.316 This observation provided the molecular explanation for the cellular defect. In 1985, the extent of clinical severity and magnitude of the cellular abnormalities were correlated with the degree of CD11/CD18 deficiency, thereby laying the groundwork for the direct relationship between the glycoprotein deficiency and the clinical presentation.315
Etiology and Pathogenesis Each of these molecules contains an α and a β subunit noncovalently associated in an αβ structure. They all have the same β subunit and are distinguished by their α subunits, which have different isoelectric points, molecular weights, and cell distribution (see Table 66–3).315 The structure of CD11/CD18 has been deduced from molecular cloning of the various subunits.315 The x-ray crystal structure and nuclear magnetic resonance analysis also reveal that activation signals lead to the separation of the α and β subunit cytoplasmic tails, thereby converting the bent conformation of each integrin with its headpiece near the plasma membrane into fully extended high-affinity structures in a switchblade-like movement.317 These studies establish that the CD11/CD18 heterodimers are members of a large gene family involved in cell–cell and cell–matrix adhesion (integrins). Several subfamilies of integrins are described and classified according to the type of their highly homologous β subunits. The α subunits are also homologous to each other, but to a lesser degree than are the associated β subunits. Within each subfamily, a single β subunit usually is shared by several α subunits. Certain α subunits often share more than one β subunit, which alters their specificity for various ligands.315 The molecular defect involves all four members of the CD11 integrin subfamily. In patients with LAD-1 who have been evaluated at the molecular level, absent, diminished, or structurally abnormal β subunits (CD18) were identified.315 A heterogeneous group of mutations that are confined to the gene on chromosome 21q22.3 also was identified.315 Many patients have point mutations that result in single amino acid substitutions in CD18, which predominantly reside between amino acids 111 and 361.315 This peptide domain is highly conserved among all β subunits and appears to be important for interaction with the α subunit. Several affected individuals are compound heterozygotes for two different mutant alleles, whereas others are homozygotes for a single mutant allele. Messenger RNA splicing abnormalities that have been described in two kindreds can result in either deletion or insertion of amino acids in the conserved extracellular domain of CD18. Small deletions within the coding sequences of the CD18 gene disrupting the reading frame or a nucleotide substitution resulting in a premature termination signal has been described. Mutations in CD18 disrupt the association in the αβ subunits so that maturation, intracellular transport, and all cell surface assembly of functionally active αβ molecules fail to occur.315 Approximately half of patients exhibit a low level of CD11/CD18 cell surface molecules and moderate disease, with the remainder having totally absent surface expression of these proteins, which accounts for a profound impairment of neutrophil and monocyte adherence and adhesion-dependent functions in vitro, including cell migration, phagocytosis, and complement- or antibody-dependent cytotoxicity.315,316
The bulk of the neutrophil Mac-1 glycoprotein is stored inside the cell in the membrane of neutrophil specific and gelatinase granules and in secretory vesicles.32,318 Exposure of neutrophils to degranulating stimuli results in a 5- to 10-fold increase in the number of Mac-1 molecules on the cell surface, which parallels the fusion of granules to the plasma membrane.318 Neutrophils from these patients fail to augment their surface adhesive glycoproteins, as the defect in β-subunit synthesis affects both membrane and granule pools of Mac-1.319 In contrast to Mac-1 and p150,95, LFA-1 is predominantly confined to the neutrophil plasma membrane. Consequently, the cell surface levels of LFA-1 are not enhanced by neutrophil degranulation.
Lymphocytes deficient in CD11/CD18 are able to adhere to endothelial surfaces via the expression on lymphocytes of very-late antigen-4 (VLA-4) integrin (synonym: integrin α4β1) receptors, which bind to the vascular cell adhesion molecule 1 (VCAM-1), found on the endothelial cells.320 This residual adhesion may account for the paucity of clinical symptoms related to lymphocyte function. The patients are not unusually susceptible to viral infection, although three patients had one or more episodes of aseptic meningitis.315
The failure of the LAD-1 neutrophils to migrate to the sites of inflammation outside of the lung and peritoneum arises from their inability to adhere firmly to surfaces and undergo transendothelial migration from venules.321,322,323 Failure of Mac-1–deficient neutrophils to undergo transendothelial migration occurs because β2 integrins bind to ICAM-1 (CD54) and ICAM-2 expressed on inflamed endothelial cells.309,324 LAD-1 neutrophils are able to accumulate in the lung, perhaps through a process of movement mediated by “chimneying,” which does not require functional integrins.325 Chemotaxis that occurs despite blockade of CD11/CD18 under special in vitro conditions has been dubbed “chimneying.” The neutrophils that do arrive at inflammatory sites in the inflamed lung by CD11/CD18-independent processes fail to recognize microorganisms coated with the opsonic complement fragment C3bi (an important stable opsonin formed by the cleavage of C3b by C3b inactivator).313,326 Other neutrophil functions, such as degranulation and oxidative metabolism, normally triggered by C3bi binding are also diminished and markedly compromised in neutrophils from LAD-1.309 Similarly, the urokinase-plasminogen activator-receptor and the FcγRIII receptors, both phosphatidylinositol-linked proteins, are defective in their functions because these receptors transduce their signals through CD11/CD18.234,327 Monocyte function is also impaired. Monocytes of affected individuals have poor fibrinogen-binding function, an activity promoted by the CD11/CD18 complex309,328; consequently, such cells are not able to participate effectively in wound healing. Thus, impairment in neutrophil function underlies the propensity to recurrent infections, which is the clinical expression of this disease. Similar genetic syndromes have been discovered in Irish Setter dogs and Holstein cattle.315 A CD11/CD18-deficient mouse with 2 to 6 percent of normal β2-integrin expression has been produced by gene targeting.321,329
Clinical Features Activated leukocytes of patients with the most-severe clinical form express less than 0.3 percent of the normal amount of the β2 integrins, whereas those of patients with the moderate phenotype may express 2 to 7 percent of normal numbers of β2-integrin molecules.309 The severely affected patients suffer from recurrent and chronic or even gangrenous soft-tissue infections (subcutaneous tissues or mucous membranes), generally by bacterial or fungal microorganisms such as S. aureus, Pseudomonas spp. and other Gram-negative enteric rods, or Candida spp. Patients with the moderate phenotype have fewer and less-severe infections. Infectious susceptibility and impaired wound healing are related to diminished or delayed infiltration of neutrophils and monocytes into extravascular inflammatory sites. In all patients surviving infancy, severe progressive generalized periodontitis is present. Individuals who are clinically well, but who are heterozygous carriers of LAD have been identified. Their stimulated neutrophils express approximately 50 percent of the normal amount of the Mac-1 α subunit and the common β subunit.309 The diagnosis of LAD-1 should be considered in infants with a paucity of neutrophils at sites of infection despite blood neutrophilia and have a history of delayed separation of the umbilical cord.
Laboratory Features The diagnosis is made most readily by flow cytometric measurement of surface CD11b in stimulated and unstimulated neutrophils using monoclonal antibodies directed against CD11b (Fig. 66–5). Assessment of neutrophil and monocyte adherence, aggregation, chemotaxis, C3bi-mediated phagocytosis, and cytotoxicity generally demonstrates striking abnormalities that are directly related to the molecular deficiency. Delayed-type hypersensitivity reactions are normal, and most individuals have normal specific antibody synthesis. The ability of lymphocytes to generate specific antibodies explains the self-limited course of varicella or viral respiratory infections. However, some patients have impaired T-lymphocyte–dependent antibody responses, for example, to repeat vaccination with tetanus toxoid, diphtheria toxoid, and polio virus.
Specific diagnosis of CD11/CD18 glycoprotein deficiency by indirect immunofluorescence flow cytometric analysis. Blood neutrophils of a pediatric patient suspected of having CD11/CD18 glycoprotein deficiency and those of an abnormal individual were subjected to immunofluorescence staining for the expression of the CD11b, CD11a, CD11c, and CD18 epitope (crosshatched histograms) as compared with the background immunofluorescence staining by isotype-identical negative-control antibodies (open histograms). Neutrophils were either stained immediately after purification by Ficoll-Hypaque density centrifugation (unstimulated) or after exposure to calcium ionophore A23187 (1 mM) for 15 minutes at 37°C (A23187-stimulated). A23187 stimulation causes significant increase in CD11b and CD18 epitope staining (surface MO1 expression) by normal neutrophils as compared with unstimulated normal cells. A23187 stimulation also causes a small increase in the CD11b-epitope expression of patient cells (the CD11b crosshatched histogram becomes distinguishable from background staining after A23187 stimulation), suggesting that this patient has a “moderate” form of the disorder (capable of expressing small but detectable quantities of CD11/CD18 glycoproteins). Flow cytometric analysis was performed on a Coulter Electronics EPICS F C Flow Cytometer with a logarithmic amplifier. (Reproduced with permission from Todd R, Freyer DR: The CD11/CD18 leukocyte glycoprotein deficiency, Hematol Oncol Clin North Am 1988 Mar;2(1):13-31.)
Patients with LAD-1 usually have blood neutrophil counts of 15 to 60 × 109/L. However, during infectious episodes, they commonly have neutrophil counts in excess of 100 × 109/L and sometimes as high as 160 × 109/L. Granulocytic hyperplasia is a feature of the marrow examination which may relate to excessive production of IL-17 and G-CSF as a result of decreased uptake of apoptotic neutrophils by tissue macrophages.319,330 Despite elevated blood counts, there is a paucity of neutrophils in inflammatory skin windows and biopsies of infected tissues.
Differential Diagnosis Eight patients (four Arab, two Turkish, one Pakistani, one Brazilian) who had neutrophilia, recurrent bacterial infections, and an inability to form pus have been described.13,331,332 The patients also had the Bombay blood phenotype (deficiency in H blood group integrins), severe mental retardation, unusual facial appearance, microcephaly, cortical atrophy, seizures, hypotonia, and short stature (see Table 66–2). Functionally, the neutrophils were unable to adhere to E-selectin or cytokine-activated endothelial cells and exhibited impaired chemotaxis and an inability to roll on postcapillary venules in vivo. The patients are now classified as having LAD-2 or congenital disorder of glycosylation type IIc (CDG-IIc).333 In contrast to LAD-1, the patients’ NK cell activity is normal. The LAD-2 neutrophils express normal levels of CD18 integrins, but are deficient in the carbohydrate structure sLex, which renders the cells unable to roll on activated endothelial cells expressing E-selectin (see Table 66–3). Thus, the neutrophils from the patients categorized as having a LAD-2 phenotype are unable to tether to inflamed venules, which is necessary for subsequent activation (Chap. 19). The LAD-2 can be explained by a congenital disorder of fucosylation of ligands for selectins and other glycoconjugates. Each of the three selectins binds with variable affinity to sialylated and fucosylated oligosaccharides, including sLex, which is present on multiple specific glycolipids and glycoproteins on leukocytes and activated endothelial cells.13 Neutrophils from LAD-2 subjects lack sLex, which leads to impaired neutrophil rolling on endothelial cells. Other fucosylated determinants, including the H, Lewis, and secretor blood group antigens, are lacking as well, suggesting a global defect in fucosylation. The diminished fucosylation arises from impaired transport of GDP-fucose from the cytoplasm to the Golgi lumen.331 A human GDP-fucose transporter (GFTP) that localizes to the Golgi apparatus has been demonstrated to be defective secondary to distinct mutations in the SLC35C1 gene encoding the transporter.13 When fibroblasts and lymphoblastoid cells derived from a LAD-2 patient were grown in the presence millimolar concentrations of fucose, cell-surface fucosylation could be restored. Following this observation oral administration of L-fucose to two Turkish patients led to normalization of neutrophil counts and functional E- and P-selectin ligands on myeloid cells accompanied by abatement of fevers and infections.13 Two Arab patients, in contrast to the Turkish patients who have different mutations of the gene encoding the putative GFTP, did not respond to oral fucose.332 A Brazilian LAD-2 patient, like the Turkish patients, initially benefited from oral fucose; but, following expression of sLex on the myeloid cells, the patient developed autoimmune neutropenia.334 The diagnosis of LAD-2 can be made by flow cytometry analysis of CD15s (sLex) expression.
LAD-3, also known as LAD-1 variant syndrome, compromises two major hallmarks: a moderate LAD-1–like syndrome and severe Glanzmann-like bleeding diathesis (Chap. 120). Four patients have been described in whom the inheritance appears to be autosomal recessive and is associated with functional defects of the leukocyte and platelet integrins arising from intracellular signaling.335 The disease initially presents in early childhood and consists of the inability to form pus at sites of microbial infections, as well as a severe bleeding tendency. The neutrophils from the patients display defective adhesion and chemotaxis and are unable to undergo the respiratory burst when triggered by unopsonized zymosan. The molecular basis for LAD-3 arises from mutations in FERMT3, which encodes kindlin-3 in hematopoietic cells. Kindlin-3 binds to regions of the β-integrin tails and constitutes an essential element for transition of integrins from the bent and inactive to the extended an active conformation.336 Marrow transplantation can be curative.
Another rare cause of neutrophilia and an inability to form pus was observed in a patient with a mutation in the Rac2 GTPase, which is discussed below. The neutrophils from the patient had defects in both adhesion and chemotaxis (see Table 66–2).
Therapy, Course, and Prognosis Treatment of LAD-1 is largely supportive.309,315 Patients with a history of recurrent infections can be maintained on prophylactic trimethoprim-sulfamethoxazole. Marrow transplantation with human leukocyte antigen (HLA)-compatible siblings or parental donors has resulted in engraftment and restoration of neutrophil function and remains the treatment of choice for patients with a severe phenotype.337
The restoration of CD11/CD18 expression in CD34 peripheral stem cells from LAD-1 following transduction with a retrovirus bearing CD18 and induced to differentiate into neutrophils with growth factors indicates that LAD-1 is caused by a defective CD18 gene and provides a basis for somatic gene therapy, which was accomplished in a dog model.338,339 Not only did the neutrophils express the integrins, but the cells demonstrated improvement in their functional responses, such as adhesion and the respiratory burst when challenged with ligands for CD11/CD18. These results indicate that ex vivo of the transfer gene for CD18 into LAD-1 CD34+ cells followed by reinfusion of the transfused cells may represent a therapeutic approach for LAD.
The severity of infectious complications correlates with the degree of β2 deficiency. Patients with severe deficiency may die in infancy, and those surviving infancy have a susceptibility to severe, life-threatening, systemic infections. In patients with moderate deficiency, life-threatening infections are infrequent and survival relatively long.319 LAD-1 can be diagnosed by prenatal screening.
Neutrophil Actin Dysfunction
These patients, like patients with LAD, have recurrent pyogenic infections from birth as a result of defective chemotactic and phagocytic response (see Table 66–2). In one patient, actin isolated from blood and neutrophils could not polymerize under conditions that fully polymerized the actin of neutrophils from normal individuals.340 Subsequent studies on the index patient’s family confirmed that partial actin dysfunction was present in the parents and one sister.341 One of the parents was found to be a heterozygote for LAD, and the other was not, but further studies established that LAD was not generally associated with defective actin filament assembly.342,343 The basis of the defective polymerization of actin in the index patient remains unknown, but this disorder of phagocytes is distinct from LAD.
Defective actin polymerization has been described in a 2-month-old infant with severe recurrent bacterial infections associated with impaired chemotaxis and phagocytic response.344 The patient’s neutrophils showed increased expression of CD11b, distinguishing the patient’s clinical problem from LAD-1. Morphologically, the neutrophils displayed thin, filamentous projections of membrane with an underlying abnormal cytoskeletal structure. Subsequently, a 47-kDa protein was purified that inhibited actin polymerization in vitro.345 Further biochemical studies revealed a markedly defective actin polymerization in the patient’s neutrophils along with a severe deficiency of an 89-kDa protein and an elevated level of the 47-kDa protein. The 47-kDa protein was identified as LSP-1 (lymphocyte-specific protein-1), which is an actin-binding protein present in normal neutrophils. Overexpression of LSP-1 resulted in bundling of actin in cells, leading to an abnormal cytoskeletal structure and motility defects.346 Neutrophils from the patient’s parents revealed a partial defect in actin polymerization accompanied by intermediate levels of LSP-1 and the 89-kDa protein. These observations suggest that the neutrophil actin dysfunction (NAD) known as NAD47/89 is an autosomal recessive disorder. Because actin dysfunction is lethal, treatment requires restoration of normal neutrophil function by marrow replacement from a normal donor. Marrow transplantation was successful.347,344
DISORDERS OF NEUTROPHIL MOTILITY
Familial Mediterranean Fever
Definition and History Familial Mediterranean fever (FMF) is an autosomal recessive disease that primarily affects populations surrounding the Mediterranean basin. The disease is characterized by acute limited attacks of fever often accompanied by pleuritis, peritonitis, arthritis, pericarditis, inflammation of the tunica vaginalis of the testes, and erysipelas-like skin disease (see Table 66–2). The initial description occurred in 1908, identifying a Jewish girl who had episodic abdominal pain and fever.348 Subsequently additional cases were identified,349 but it took nearly a half century to establish this disorder as familial Mediterranean fever.350
Epidemiology More than 10,000 patients worldwide are affected with FMF. It occurs predominantly in Sephardic Jews, Arabs, Turks, Italians, and Armenians.348 The disorder can occur in other populations, but it is unusual. The frequency of the susceptibility gene varies widely; it is very high among Armenians (ratio of persons with the gene to those without it is 1:7) and Sephardic Jews (1:5 to 1:16), but is lower in Ashkenazi Jews (1:135).
Etiology and Pathogenesis The pathologic findings in FMF are those of nonspecific acute inflammation affecting serosal tissues such as the pleura, peritoneum, and synovium. Neutrophilic infiltration predominates in the affected tissues. Physical and emotional stress, menstruation, and a high-fat diet may trigger the attacks.351
The gene responsible for FMF has been identified to be located on chromosome 16. It encodes for a 781-amino-acid protein called pyrin or marenostrin.352 The gene (MEFV) is predominantly expressed in neutrophils, eosinophils, monocytes, dendritic cells, and synovial and peritoneal fibroblasts, and its expression is upregulated by IFN-γ and TNF, and by the process of myeloid differentiation itself.353 Nearly all the 50 mutations in the MEFV gene are missense changes, most of which are clustered in on exon 2 and 10.354 Founder effects in FMF have been established, and the two most common mutations, V726A and M694V, originated in common ancestors who lived about 2500 years ago in the Middle East.352
Pyrin plays a role in controlling the activity of inflammasomes (see “Neutrophil Surface Receptors”). PYRIN, one of the four domains of pyrin, bears homology to a number of proteins involved in apoptosis and in inflammation, and is similar to a member of the six-helix-bundle death-domain superfamily that includes death domains and death effector domains known as CARDs.355 The PYD appears to allow for the interaction of macromolecular complexes by PYRIN–PYRIN interactions. This interaction has led to the identification of pyrin’s ability to interact specifically with another PYRIN-domain protein termed apoptosis-associated speck-like protein with a CARD (ASC).356 Besides the aminoterminal PYD, ASC has a C-terminal CARD domain that allows binding to the CARD of procaspase-1 (IL-1β–converting enzyme), which results in procaspase-1 autoactivation.355 Activated caspase-1 then converts prointerleukin-1β to IL-1β, which is, in turn, secreted and interacts with the IL-1 receptor to mediate inflammation. It has been suggested that pyrin may act as an antiinflammatory molecule by inhibiting ASC-induced IL-1 processing, which, in turn, could be defective in FMF. This hypothesis is supported by observing increased IL-1 processing and heightened sensitivity to LPS and impaired apoptosis in peritoneal macrophages from pyrin knockout mice. The puzzle, however, remains as to why serosal tissues are the main targets of inflammation in FMF.
Clinical Features Febrile episodes in FMF may begin in infancy, but by age 20 years, 90 percent of patients have had their first attack. The duration and frequency of attacks may vary considerably, even in the same patient.351 Acute attacks frequently last 24 to 48 hours and recur once or twice a month. In some patients, attacks may recur as frequently as several times a week, or as infrequently as once a year, and symptoms may persist as long as a week during individual episodes. Some patients experience spontaneous remission that persists for years, followed by recurrence of frequent attacks. Peritonitis caused by FMF may resemble an acute abdomen, thereby leading to potential uncertainties about the clinical management of the acute abdominal episode. Attacks of pleuritic pain occur in approximately 25 to 80 percent of patients. Symptoms of pleuritis may sometimes precede abdominal pain, and some patients experience pleuritic attacks without abdominal symptoms. Recurrent pericarditis is rare. The course of peritonitis in FMF is similar to attacks at other serosal sites; however, it tends to appear at a late stage of the disease. Mild arthralgia is a common feature of febrile attacks, and monoarticular or oligoarticular arthritis may occur. Arthritis usually affects large joints, the knees in particular, and effusions are common. As many as one-third of the patients experience transient erysipelas-like skin lesions that appear typically on the lower leg, ankle, or dorsum of the foot. These lesions are circumscribed, painful, erythematous areas of swelling, which usually subsides within 24 to 48 hours.
In approximately 25 percent of affected patients, a form of renal amyloidosis develops in which the amyloid derives from a normal serum protein called serum amyloid A (amyloidosis of the AA type; Chap. 108). The amyloidosis progresses over a period of years to renal failure in almost all cases, and the cause of death in patients with FMF is usually attributed to this complication. It appears that polymorphisms in the gene for serum amyloid A increase the susceptibility to renal amyloidosis and that polymorphisms in a gene for the major histocompatibility complex class 1 α-chain influence the severity of the disease.350
Laboratory Features Laboratory findings in FMF are nonspecific. Nonspecific findings include increases in inflammatory mediators such as amyloid A, fibrinogen, and C-reactive protein during febrile attacks.350 Proteinuria greater than 0.5 g of protein per 24 hours in patients with FMF may suggest amyloidosis.
The cloning of the FMF gene now allows a reliable diagnostic test. Five founder mutations account for 74 percent of FMF carrier chromosomes from typical populations known to harbor the disease.357 Carrier rates for FMF mutations may be as high as 1:3 in some populations, suggesting that the disease is often underdiagnosed. Some amino acids that cause human disease are often present in wild-type in primates.358
Differential Diagnosis The TNF receptor–associated periodic syndrome (TRAPS) was first described in 1982 in a large Irish family.359 The affected family members had recurrent fever with localized myalgia and painful erythema. Differentiating this disorder from FMF was its response to corticosteroids and the autosomal dominant inheritance of the disorder. Affected patients can have attacks that last for at least 1 or 2 days, but prolonged attacks lasting longer than a week are common. Localized pain and tightness in one muscle group and a migratory pattern of the symptoms are prominent features. The disorder may be associated with colicky abdominal pain, diarrhea or constipation, nausea, and or vomiting. Painful conjunctivitis, periorbital edema, or both are common as well as chest pain secondary to sterile pleuritis.350 During febrile attacks, painless skin lesions may develop on the trunk or extremities and may migrate distally. Missense mutations in the gene for the type-1 TNF 55-kDa cell membrane receptor, which is required for diagnosis, have been identified. Patients with TRAPS respond dramatically to high doses of oral prednisone (>20 mg). In time, however, the responses wane, requiring higher doses of corticosteroids. Standard doses of a p75:Fc fusion protein, etanercept, administrated subcutaneously twice weekly decreases the frequency, duration, and severity of attacks; thus, etanercept may provide a safer, more effective alternative then corticosteroids in controlling the disease.
Therapy Colchicine treatment is effective in FMF and may prevent the development of amyloidosis.351 Prophylactic colchicine, 0.6 mg orally, two to three times a day, prevents or substantially reduces the acute attacks of FMF in most patients. Some patients can abort attack with intermittent doses of colchicine beginning at the onset of attacks (0.6 mg orally every hour for 4 hours, then every 2 hours for four doses, and then every 12 hours for 2 days). In general, patients who benefit from intermittent colchicine therapy are those who experience a recognizable prodrome before developing fever and clear-cut acute symptoms.
Course and Prognosis The prognosis for normal longevity for patients has been excellent since the recognition that colchicine is an effective treatment of this disease. Most patients can be maintained almost entirely symptom-free. However, if amyloidosis develops, it may be followed by the nephrotic syndrome or uremia. Unless the patient receives a renal transplant, the likelihood of eventual death from renal failure is high.
Other Disorders of Neutrophil Motility
The directed migration of neutrophils from the circulation to an inflammatory site is a consequence of chemotaxis and leads to the accumulation of an exudate. For normal chemotaxis to occur, a complex series of events must be coordinated. Chemotactic factors must be generated in sufficient quantities to establish a chemotactic gradient. The neutrophils must have receptors for the chemotactic agents and mechanisms for discerning the direction of the chemotactic gradient. Depressed neutrophil chemotaxis has been observed in a wide variety of clinical conditions (see Table 66–2).360 These can be stratified as follows: (1) defects in the generation of chemotactic signals; (2) intrinsic defects of the neutrophil; and (3) direct inhibitors of neutrophil motility in response to chemotactic factors.
Older patients with chemotactic disorders may be infected by a variety of microorganisms, including fungi and Gram-positive or Gram-negative bacteria. S. aureus is the most frequent bacterial offender. Typically, the skin, gingival mucosa, and regional lymph nodes are involved. Respiratory tract infections are frequent, but sepsis is rare. Delayed or inappropriate signs and symptoms of inflammation are common. Although the cells move slowly in Boyden chambers or other chemotactic assays, they do accumulate in sufficient numbers in inflammatory sites to produce pus. However, detection of patients with neutrophils that have profound defects in chemotaxis usually is accomplished through other phagocytic assays.
Patients with the hereditary deficiency of complement factors C3, C5, or properidin exhibit an increased incidence of bacterial infections because they are unable to form the chemotactic peptide C5a.361 The degree to which defective chemotaxis plays a role in C3 deficiency is unclear because opsonization and ingestion rates also are abnormal in these disorders. Frequently, chemotactic disorders are associated with other impaired neutrophil functions. For instance, both glycogen storage disease type 1b362 and Shwachman-Diamond syndrome363 are chemotactic disorders frequently associated with an absolute neutrophil count below 0.5 × 109/L. Following restoration of a normal neutrophil count with G-CSF, the patients no longer are predisposed to recurrent bacterial infections in spite of a persistent chemotactic defect. Thus, a chemotactic defect observed in vitro does not correlate invariably with decreased resistance to bacterial infections in vivo.
Among the impaired defense mechanisms of the neonate is neutrophil adherence and chemotaxis, as demonstrated by the in vitro response of neonatal neutrophils to a variety of chemotactic factors.322 The impaired motility of the neonatal neutrophils in part arises from the diminished ability to mobilize neutrophil β2 integrins following neutrophil activation.364 Additionally, the neonatal neutrophil may have a qualitative defect in β2-integrin function, resulting in impaired neutrophil transendothelial migration for up to 1 month after birth.
At the other end of the spectrum, neutrophils from elderly loose focus during chemotaxis while their motility is unimpaired. This is caused by increased activity of PI3-K and results in less efficient bacterial killing and enhanced release of tissue destructive proteases. Inhibition of PI3K activity reverts this condition in vitro.365
Drugs and Extrinsic Agents That Impair Neutrophil Motility
Although many pharmacologic agents can influence neutrophil function, few drugs used in clinical medicine affect neutrophil behavior in vivo. Ethanol, an inhibitor of PLD, in concentrations that occur in human blood can inhibit neutrophil locomotion and ingestion.366 Glucocorticoids, especially at high and sustained doses, inhibit neutrophil locomotion, ingestion, and degranulation.367 Administration of glucocorticoids on alternate days does not interfere with neutrophil movement.368 Epinephrine does not have a direct effect on neutrophil adhesion but cyclic adenosine monophosphate (cAMP), which is released from endothelial cells following exposure to epinephrine, can depress neutrophil adherence.369 Similarly, elevated cAMP levels following epinephrine administration may impair neutrophil adherence, leading to diminished neutrophil margination and apparent neutrophilia. Immune complexes, as seen in patients with rheumatoid arthritis or other autoimmune diseases, also can inhibit neutrophil movement by binding to neutrophil Fc receptors.
Hyperimmunoglobulin E Syndrome
Definition and History Autosomal dominant hyperimmunoglobulin E syndrome (HIES) is a disorder characterized by markedly elevated serum IgE levels, chronic dermatitis, and serious recurrent bacterial infections.370 The skin infections in these patients are remarkable for their absence of surrounding erythema, leading to the formation of “cold abscesses.” The neutrophils and monocytes from patients with this syndrome exhibit a variable, but at times profound, chemotactic defect that appears extrinsic to the neutrophil (see Table 66–2).371
The syndrome was originally described in 1966 in two red-headed, fair-skinned females who had “cold abscesses” and hyperextensible joints, which led to the appellation “Job’s syndrome.”370 Subsequently Buckley and coworkers documented the association of levels of immunoglobulin E with undue susceptibility to infection.372
Epidemiology Reports of more than 200 cases have been documented.372,373 HIES occurs in persons from diverse ethnic backgrounds and does not seem to be more common in any specific population.
Etiology and Pathogenesis Both males and females have been affected, as well as members of succeeding generations, indicating that the disorder is autosomal dominant with an incomplete penetrance form of inheritance.370 STAT3 mutations cause most, if not all cases of autosomal dominant HIES. All mutations have been missense mutations or in-frame deletions, leading to the formation of full-length mutant STAT3 protein, which exerts a dominant negative effect. STAT3 is a major transduction protein affecting pathways involving wound healing angiogenesis, immunity, and cancer. The more rare autosomal recessive form is caused by mutations in dedicator of cytokinesis 8 (DOCK8), a guanine nucleotide exchange factor.374
The mechanism of the immune deficiencies in HIES remains clouded. Several reports with limited numbers of patients have conflicted results as to whether a chemotactic defect exists and whether there is a T-helper 1/T-helper 2 cytokine imbalance.
Clinical Features HIES may begin as early as day 1 after birth.372 The syndrome is characterized by chronic eczematoid rashes, which are typically papular and pruritic. The rash generally involves the face and extensor surfaces of arms and legs; skin lesions are frequently sharply demarcated and usually lack surrounding erythema. By 5 years of age all patients have had a history of recurrent skin abscess formation with recurrent pneumonias, along with chronic otitis media and sinusitis. Patients may also develop septic arthritis, cellulitis, or osteomyelitis. The major offending pathogen is generally S. aureus. Other pathogens commonly infecting patients are C. albicans, H. influenzae, and pneumococci. Other associated features include coarse facial features, including a prominent forehead, deep set eyes, a broad nasal bridge, a wide fleshly nasal tip, mild prognathism facial asymmetry, and hemihypertrophy.370 There is a high incidence of scoliosis, hyperextensible joints, and delayed shedding of the primary teeth.370 Occasionally, unexplained osteopenia presents, which is often complicated by recurrent bone fractures. Additionally, there is an increased risk of both Hodgkin and non-Hodgkin lymphoma.
Laboratory Features Blood and sputum eosinophilia have been a consistent finding in all patients.370 Patient serum IgE levels range from three to 80 times the upper limit of normal. The serum IgE usually rises above 2000 IU/mL and often is elevated at birth. Upon reaching adulthood the IgE may decline over years, despite the clinical abnormalities of STAT3 deficiency. Usually patients have normal concentrations of IgG, IgA, and IgM, and may have elevated levels of IgD. Patients often have abnormally low anamnestic antibody response and poor antibody and cell-mediated responses to neoantigens. At times the neutrophils and monocytes of patients have a profound chemotactic defect.
Differential Diagnosis Autosomal recessive-HIES (AR-HIES) is a distinct clinical entity manifested by elevated IgE ligands, and recurrent skin and cutaneous viral infections and mutations in DOCK8.370,375
Fatal sepsis occurs in AR-HIES from both Gram-positive and Gram-negative bacteria. Patients with AR-HIES have more symptomatic neurologic disease than STAT3 deficiency. Autoimmune hemolytic anemia may occur, but neutrophil chemotaxis is normal. The genetic mutation underlying AR-HIES remain unclear. Therapy remains supportive.
Therapy No known therapy is curative, and management decisions are based on the clinical findings. Prophylactic trimethoprim-sulfamethoxazole is effective in reducing infections with S. aureus.370 Type and route of antibiotic therapy are dictated by the results of the Gram stain and culture in patients with acute bacterial infections. Incision and drainage are essential for the management of abscesses, including superinfected pneumatoceles. Eczematoid dermatitis can be controlled with topical glucocorticoids to reduce inflammation and antihistamines to control pruritus. Intravenous immunoglobulin may decrease the number of infections for some patients. Attention needs to be paid to the scoliosis, fractures and degenerative joints by orthopedists. Retention of primary teeth requires dental expertise.
Course and Prognosis If the hyperimmunoglobulin E is recognized early in life and the patient is maintained on chronic anti-Staphylococcal antibiotic therapy, the prognosis remains good. Many such patients have reached maturity, indicating that the syndrome is compatible with prolonged survival. Conversely, if the diagnosis is delayed and the patient develops infected giant pneumatoceles, secondary fungal infections may occur, leading to a morbid state.
DEFECTS IN MICROBICIDAL ACTIVITY
Chronic Granulomatous Disease
Definition and History CGD is a genetic disorder affecting the function of neutrophils and monocytes. These phagocytic cells are able to ingest, but not kill, catalase-positive microorganisms because of an inability to generate antimicrobial oxygen metabolites (see Table 66–2). It is caused by mutations involving one of several genes encoding a component of the NADPH oxidase.376
In 1957, two pediatric groups caring for six male infants reported a clinical disorder of chronic suppurative lymphadenitis and recurrent fevers leading to premature deaths in the children.377,378 In the same time period, three observations assisted in providing the framework to understand the defect in the phagocytes of patients with CGD. Scientists described first that a striking increase in oxygen consumption was found upon particle ingestion by phagocytes, which was not related to mitochondrial oxygen metabolism.379 Next, it was found that the process of phagocytosis was accompanied by the formation of large quantities of H2O2 in the cell.380 Subsequently, it was reported that homogenates of phagocytes consume oxygen when incubated with pyridine nucleotides.381 These observations indicated that an oxidase enzyme or enzymes in the phagocytes were activated during phagocytosis to convert molecular oxygen into H2O2. It was then established that phagocytes from patients with CGD could ingest, but could not kill, the catalase-positive organisms.381 Building on previous studies that a neutrophil oxidase mediates the increase in oxygen consumption, a pyridine-dependent oxidase was found to be deficient in neutrophils of patients with CGD, which led to their inability to reduce the dye nitroblue tetrazolium (NBT) during phagocytosis of particles.382 Collectively, these studies laid the groundwork for subsequent studies to unravel the biochemical and genetic defects in CGD.
Epidemiology The incidence of CGD in the United States is 1 per 200,000 livebirths, based on data from the National Institutes of Allergy and Infectious Disease Registry.383 Data from the Registry indicates that 86 percent of patients are male and 14 percent female; 80 percent are classified as white, 11 percent as black patients, and 3 percent Asians or mixed-race patients. Of the 340 patients in the Registry with adequate information for determination genetic transmission, 70 percent had the X-linked recessive form of the disease.
Etiology and Pathogenesis Several laboratory tests are used to classify forms of CGD and aid in understanding its pathogenesis (Table 66–4). The diagnosis of CGD is based on a compatible clinical history and demonstration of a defective respiratory burst. Several methods detect the production of reactive oxidants. The NBT method relies on the intracellular reduction of NBT by superoxide anion to a blue formazan precipitate that can be seen microscopically.376 More sensitive methods rely on the reaction of oxidants with specific chemiluminescent and fluorescent probes. The patients with CGD may have heterogeneous array of regular symptoms and severity, depending on which subunit is defective and on the nature of the genetic mutation.
Table 66–4.Diagnostic Classification of Chronic Granulomatous Disease ||Download (.pdf) Table 66–4. Diagnostic Classification of Chronic Granulomatous Disease
|Affected Component ||Inheritance ||Subtype ||Membrane-Bound Cytochrome b558* ||Cytosol p47phox* ||Cytosol p67phox* |
|gp91phox ||X ||X910 ||Not detectable ||Normal ||Normal |
| || ||X91+ ||Normal quantity, but nonfunctional ||Normal ||Normal |
| || ||X91– ||Defective gp91phox, which is poorly functional or expressed in a small fraction of phagocytes ||Normal ||Normal |
|p22phox ||A ||A220 ||Not detectable ||Normal ||Normal |
| || ||A22+ ||Normal quantity, but nonfunctional ||Normal ||Normal |
|p47phox ||A ||A470 ||Normal quantity ||Not detectable ||Normal |
|p67phox || ||A670 ||Normal ||Normal ||Not detectable |
Nicotinamide Adenine Dinucleotide Phosphate-Oxidase Function Engulfment of microbes by phagocytic cells is associated with a burst of oxygen consumption that is important for microbicidal killing and digestion. The respiratory burst is accompanied, not by mitochondrial respiration, but by a unique electron transport chain called the NADPH oxidase. Prior to stimulation, the components of the oxidase are physically separated into two major subcellular locations (Fig. 66–6). The membrane-bound portion of the NADPH oxidase contains a heterodimeric cytochrome b558 composed of a large, heavily glycosylated subunit with a Mr of 91 kDa, known as a gp91phox (91-kDa glycoprotein of the phagocyte oxidase), and a 22-kDa protein known as p22phox.376,384 Eighty to 90 percent of the cytochrome b558 is found in specific and gelatinase granules and secretory vesicles of the neutrophil and following neutrophil activation translocates to the plasma membrane.66,318 The heavy chain of cytochrome b contains sites for heme binding, flavin adenine dinucleotide (FAD) groups, and NADPH binding.385,386,387,388 The three-dimensional structure of cytochrome b558 indicates that the carboxyl-terminal half of the peptide contains sequences for flavin and NADPH binding.389 The amino half of the molecule is hydrophobic and contains the histidines that coordinate heme binding.390 The p22phox also contains a site for heme binding.385 The synthesis of the p22phox peptide is absolutely required for stability of gp91phox and for oxidase activity in the membrane.376 The p22phox also contains proline-rich regions that display consensus protein–protein interactions that provide a binding site for p47phox.391 Three other proteins vital to the function of this oxidase system reside in the cytosol of the resting phagocyte. Upon stimulation, translocation of p47phox takes place. Phosphorylated p47phox together with two other cytoplasmic components of the oxidase, p67phox, and a low-molecular-weight guanosine triphosphate Rac-2, translocate to the membrane, where they interact with cytoplasmic domains of the transmembrane cytochrome b558 to form the active oxidase.391,392 Both p47phox and p67phox contain SH3 (Src homology 3) domains that may participate in intramolecular and intermolecular binding with consensus proline-rich regions in p47phox.392 Phosphorylation, which occurs on serines in the cationic C-terminal region of p47phox, serves to disrupt this intermolecular interaction, making the SH3 regions available for binding to p22phox. Another cytoplasmic component with homology to p47phox has been identified as p40phox. p40phox, like p47phox, contains a PX domain, a motif that supports the binding to phosphoinositides on the cytosolic side of membranes.393 The p40phox component stabilizes the cytoplasmic complexes of p67phox and p47phox on phagosomes. Its binding of phosphatidylinositol 3 phosphate also potentiates superoxide production upon neutrophil activation.394 Cytochrome b558 spans the membrane, permitting NADPH to be oxidized at the cytoplasmic surface and oxygen to be reduced to form O2– on the outer surface of the plasma membrane or on the inner surface of the phagosomal membrane.395
Possible mechanisms for the production of superoxide anion in neutrophils. Oxygen is reduced to superoxide (O2–) by an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The oxidase is a composite of (1) a 47-kDa cytosolic protein (p47); (2) a 67-kDa cytosolic protein (p67); (3) a 40-kDa cytosolic protein (p40); (4) a low-molecular-weight cytosolic G-protein, Rac2; and (5) a membrane-bound cytochrome b558. Cytochrome b consists of a 22-kDa protein subunit (p22) and a 91-kDa glycoprotein subunit (gp91), both of which contain heme. The gp91 subunit is a flavin adenine dinucleotide (FAD)-dependent flavoprotein that contains the NADPH binding site and ultimately shuttles electrons to molecular oxygen, forming O2–, and (6) the cytosol components translocate to the membrane and may serve to alter the tertiary structure of cytochrome b, to permit the flow of electrons from NADPH to O2. The p47 subunit (p47) is phosphorylated upon activation of the neutrophil. The p40phox component stabilizes the preactivation complex of p67phox. The unstable superoxide anion (O2–) is converted to hydrogen peroxide (H2O2), either spontaneously or by the enzyme superoxide dismutase (SOD). H2O2 in the presence of myeloperoxidase (MPO) converts H2O2 to hypochlorous acid (HOCl). Both H2O2 and O2– can be transformed into hydroxyl radical (OH–). H2O2 can be reduced to H2O and O2 by the enzyme catalase or by glutathione (GSH), a product of the hexose-monophosphate shunt. These reactive oxygen species are responsible for microbial killing. Normal oxidative function of the NADPH complex requires fully functional individual components.
Genetic Alterations Affecting Cytochrome b The most frequent form of CGD occurs in 70 percent of patients and is caused by mutations in the gp91phox gene, termed CYBB, which is located on chromosome Xp21.1.376,396 These mutations lead to the X-linked form of the disease. Large interstitial deletions causing other X-linked disorders such as retinitis pigmentosa, Duchenne muscular dystrophy, McLeod hemolytic anemia, and ornithine transcarbamylase deficiency, have been reported in a few patients with X-linked CGD.383,397,398,399 Mutation analysis of the gene encoding gp91 and a large group of X-linked CGD kindreds has documented many distinct defects, including point mutations, inversions, deletions, or insertions that disrupt the reading frame and nonsense mutations that create a premature stop codon.396 Some splice-site defects have also been identified. In this situation, short deletions in gp91phox mRNA are caused by point mutations that produce partial or complete exon skipping during mRNA splicing.400 This abnormality is a common cause of X-linked CGD. In the remaining patients, point mutations have been identified that generate either premature stop codons or amino acid substitutions that apparently disrupt protein stability or function and lead to a complete lack of detectable cytochrome b558 protein in phagocytic cells in most patients with X-linked CGD. In some situations, low levels of functional cytochrome b are present, whereas in others, normal levels of dysfunctional cytochrome b558 occur.401 In the latter situation there is some clustering of defects in regions of known function, such as the NADPH- or flavin-binding consensus regions.402 Approximately 10 to 15 percent of X-linked CGD arises from new germline mutations.403
A similar array of mutations has been identified in the 5 percent of CGD patients who have abnormalities in the p22phox gene, termed CYBA, which is located on chromosome 16q24.376,402,404 In this autosomal disorder, mutations in the p22phox gene result in deletions, frameshifts, and/or missense mutations. Patients with a defective p22phox gene do not express the other cytoplasmic unit polypeptide. In one patient, p22phox peptide was associated with normal amounts of cytochrome b with normal heme spectrum, but p47phox translocation membrane did not occur and there was no oxidase activation because the mutation affected a proline-rich region thought to mediate binding to one of the SH3 domains of p47phox. In gp91phox-deficient patients, p22phox mRNA is present, but it is not translated, which is consistent with the notion that either cytochrome subunit polypeptide is dependent upon the stable expression of the other subunit.376
Genetic Alterations Affecting Cytosolic Proteins Two other proteins have been identified as being vital to the function of the NADPH-oxidase system. Their absence results in the syndrome of CGD.405 These proteins have molecular masses of 47 kDa and 67 kDa, respectively, and are located in the cytosol of resting cells. Defects in the genes for p47phox, termed NCF1, which is found on chromosome 7q11, are responsible for the majority of all cases of autosomal recessive CGD, whereas inherited defects for the gene for neutrophil p67phox, termed NCF2, account for a small subgroup of autosomal recessive CGD.376 The function of p47phox and p67phox in regulating the respiratory burst oxidase is thought to involve activation of the electron transport function of cytochrome b558. The mutation analysis in patients with p47phox-deficient forms of CGD reveals an unusual pattern, in that more than 90 percent of mutant alleles have guanine-thymine dinucleotide deletion at the start of exon 2, resulting in frameshift and premature stop.402,406 The truncated protein is unstable in that it cannot be detected immunologically. The majority of patients appear to be homozygous for this mutation without any history of consanguinity. The p47phox gene occurs in an area of chromosome 7 that has a high degree of evolutionary duplication in normal individuals because a pseudogene highly homologous to the normal p47phox gene exists in the normal genome in this region of duplication. The pseudogene contains the same GT deletion associated with most cases of p47phox CGD. This implies that recombination of the normal gene and pseudogene with conversion of the normal gene to partial pseudotype sequence in that region may be responsible for the high relative rate of this specific mutation in diverse racial groups, which proved to be the case.407
A second rare form of CGD is caused by mutations in the gene for the p67phox cytosolic component.401 The p67phox gene, which has been mapped to the long arm of chromosome 1, spans 37 kb and contains 16 exons. The mutations identified in p67phox-deficiency CGD have included missense mutations and spliced junction mutations affecting mRNA processing, which led to nondetectable p67phox protein by immunologic means.402
Mutation of NCF4, the gene encoding p40phox, was reported in a child with granulomatous colitis. One allele had a frameshift mutation with a premature stop codon. The other had a missense mutation predicting an R105Q substitution in the PX domain which is responsible for binding to phosphatidylinositol 3 phosphate. The functional defect was inability to assemble the NADPH oxidase in the membrane of phagosomes but not on the plasma mambrane.408
Predisposition to Infection Mutations in the gene for cytochrome b558 or the cytosolic factors involved in activating the cytochrome are associated with the CGD phenotype. Figure 66–7 shows schematically the manner in which the metabolic deficiency of the CGD neutrophil predisposes the host to infection. Normal neutrophils accumulate H2O2 and other oxygen metabolites in the phagosomes containing ingested microorganisms. MPO is delivered to the phagosome by degranulation and in this setting H2O2 acts as a substrate for MPO to oxidize halide to HOCl and chloramines, which kill the microbes. The quantity of H2O2 produced by the normal neutrophils is sufficient to exceed the capacity of catalase, a H2O2-catabolizing enzyme produced by many aerobic microorganisms, including S. aureus, most Gram-negative enteric bacteria, C. albicans, and Aspergillus spp. In contrast, H2O2 is not produced by CGD neutrophils, and any generated by the microbes themselves may be destroyed by their own catalase. Thus, catalase-positive microbes can multiply inside CGD neutrophils, where they are protected from most circulating antibiotics, and can be transported to distant sites and released to establish new foci of infection.405 Activation of the oxidase also has a pronounced effect on the pH within the phagocytic vacuole. It is controversial whether activation of the respiratory burst is associated with an alkaline phase, but the pH of the phagocytic vacuole becomes more acidic in CGD patients than in normal patients.161,409 The alkaline phase may be important for the antimicrobial and digestive functions of the neutral hydrolases released from the cytoplasmic granules into the vacuole upon phagocytosis. In CGD, the phagocytic vacuoles remain acidic and the bacteria are not digested properly.410 The impairment in the respiratory burst by CGD neutrophils leads to delayed neutrophil apoptosis and subsequent impaired clearance of degenerating neutrophils by CGD macrophages, which, in turn, predisposes the host to enhanced inflammation.411 CGD neutrophils are incapable of generating NETs and cannot trap microorganisms by this mechanism.412 The CGD macrophage is unable to clear CGD neutrophils because of a deficiency of intrinsic IL-4 production, which occurs because of defective phosphatidylserine exposure on CGD neutrophils, that is a necessary requirement to engage CGD macrophage phosphatidylserine membrane receptors and subsequent macrophage activation.411 In hematoxylin-and-eosin-stained sections from patients, macrophages eventually may contain a golden pigment, which reflects the abnormal accumulation of ingested material and also contributes to the diffuse granulomata that give CGD its descriptive name.413 On the other hand, when CGD neutrophils ingest pneumococci or streptococci, these organisms generate enough H2O2 to result in a microbicidal effect.
The pathogenesis of chronic granulomatous disease (CGD). The manner in which the metabolic deficiency of the CGD neutrophil predisposes the host to infection is shown schematically. Normal neutrophils accumulate hydrogen peroxide (H2O2) in the phagosome containing ingested Escherichia coli. Myeloperoxidase is delivered to the phagosome by degranulation, as indicated by the closed circles, and in this setting, H2O2 acts as a substrate for myeloperoxidase to oxidize halide to hypochlorous acid and chloramines, which kill the microbes. The quantity of H2O2 produced by the normal neutrophils is sufficient to exceed the capacity of catalase, a H2O2-catabolizing enzyme of many aerobic microorganisms, including most Gram-negative enteric bacteria, Staphylococcus aureus, Candida albicans, and Aspergillus spp. When organisms such as E. coli gain entry into the CGD neutrophils, they are not exposed to H2O2 because the neutrophils do not produce it, and the H2O2 generated by microbes themselves is destroyed by their own catalase. When CGD neutrophils ingest streptococci (Strep.) or pneumococci, these organisms generate enough H2O2 to result in a microbicidal effect. On the other hand, as indicated in the middle figure, catalase-positive microbes, such as E. coli, can survive within the phagosome of the CGD neutrophil.
Clinical Features Although the clinical presentation is variable, several clinical features suggest the diagnosis of CGD.376 Any patient with recurrent lymphadenitis should be considered to have CGD. Additionally, patients with bacterial hepatic abscesses, osteomyelitis at multiple sites or in the small bones of the hands and feet, a family history of recurrent infections, or unusual catalase-positive microbial infections all require clinical evaluation for this disorder. Table 66–5 lists the most common clinical infections that afflict CGD patients and Table 66–6 cites their prevalence.
Table 66–5.Common Infecting Organisms Isolated from Chronic Granulomatous Disease Patients ||Download (.pdf) Table 66–5. Common Infecting Organisms Isolated from Chronic Granulomatous Disease Patients
|Infection Type ||Organism ||X-Linked Recessive (%) ||Autosomal Recessive (%) |
|Pneumonia ||Aspergillus spp. ||41 ||29 |
| ||Staphylococcus spp. ||11 ||13 |
| ||Burkholderia cepacia ||7 ||11 |
| ||Nocardia spp. ||6 ||13 |
| ||Serratia spp. ||4 ||5 |
|Abscess || || || |
|Subcutaneous ||Staphylococcus spp. ||28 ||21 |
| ||Serratia spp. ||19 ||9 |
| ||Aspergillus spp. ||7 ||0 |
|Liver ||Staphylococcus spp. ||52 ||52 |
| ||Serratia spp. ||6 ||4 |
| ||Candida spp. ||12 ||0 |
|Lung ||Aspergillus spp. ||27 ||18 |
|Perirectal ||Staphylococcus spp. ||9 ||15 |
|Brain ||Aspergillus spp. ||75 ||25 |
|Suppurative adenitis ||Staphylococcus spp. ||29 ||12 |
| ||Serratia spp. ||9 ||15 |
| ||Candida spp. ||7 ||4 |
|Osteomyelitis ||Serratia spp. ||32 ||12 |
| ||Aspergillus spp. ||25 ||18 |
|Bacteremia/fungemia ||Salmonella spp. ||20 ||13 |
| ||Burkholderia cepacia ||13 ||0 |
| ||Candida spp. ||9 ||25 |
| ||Staphylococcus spp. ||11 ||0 |
Table 66–6.Prevalence of Infectious Complication of Chronic Granulomatous Disease Patients ||Download (.pdf) Table 66–6. Prevalence of Infectious Complication of Chronic Granulomatous Disease Patients
|Infection Type ||X-Linked Recessive (%) ||Autosomal Recessive (%) |
|Pneumonia ||80 ||77 |
|Abscess (all) ||68 ||70 |
|Subcutaneous ||43 ||42 |
|Liver ||26 ||33 |
|Lung ||16 ||14 |
|Brain ||3 ||5 |
|Perirectal ||17 ||7 |
|Suppurative adenitis ||59 ||32 |
|Osteomyelitis ||27 ||21 |
|Bacteremia/fungemia ||21 ||10 |
|Cellulitis ||7 ||5 |
Among the various infections, only perirectal abscess, suppurative adenitis, and bacteremia/fungemia differ significantly in prevalence in the X-linked recessive and autosomal recessive CGD patients.383 Each of these conditions was twice as common in the X-linked form.
The onset of clinical signs and symptoms may occur from early infancy to young adulthood. Although the majority of patients with CGD (76 percent) are diagnosed before the age of 5 years, approximately 10 percent are not diagnosed until the second decade of life, and on rare occasions, not until the third decade or later.383 The organisms infecting CGD patients have changed considerably from those initially reported between 1957 and 1976. Staphylococcus caused most of the infections in the initial cases; Klebsiella and E. coli were then the next most common pathogens. Now Aspergillus is the prominent organism causing pneumonia and is the leading cause of death in patients.376 Invasive aspergillosis can occur in the first few months of life in healthy infants as well as in those with CGD. Although aspergillosis is the most common infecting fungus in CGD, Candida and several other fungal strains have been invasive in this disorder. Burkholderia cepacia is another leading cause of death in patients with CGD. Serratia marcescens is the third leading organism that commonly infects patients with CGD. Infections are characterized by microabscesses and granuloma formation. The presence of pigmented histiocytes is helpful in establishing the diagnosis. Patients may suffer from the consequences of chronic infections including the anemia of chronic disease, lymphadenopathy, hepatosplenomegaly, chronic purulent dermatitis, restrictive lung disease, gingivitis, hydronephrosis, and gastroenteric narrowing.383 Patients with CGD are also at risk for developing colitis and chorioretinitis, and discoid lupus erythematosus.383
Several mothers of patients in whom X-linked inheritance was established had an illness resembling systemic lupus erythematosus.383 Both X-linked and autosomal recessive patients with CGD also have a similar disorder.414 It may be that these mothers’ and patients’ cells are unable to clear immune complexes sufficiently, which is a characteristic feature of CGD cells in vitro.415 Variant alleles of MBL and FcγRIIA especially in combination are associated with rheumatologic disorders in patients with CGD.416
Laboratory Findings The defect in the respiratory burst is best determined by measuring superoxide or H2O2 production in response to both soluble and particulate stimuli.417 A test that is being employed is the use of flow cytometry using dihydrorhodamine-123 fluorescence.418 Dihydrorhodamine-123 fluorescence detects oxidant production because it increases fluorescence upon oxidation.418 In most cases there is no detectable superoxide or H2O2 generation with either type of stimulus. In the variant form of CGD, however, superoxide may be produced at rates between 0.5 and 10 percent of control.419
An alternative method for measuring respiratory burst activity is the NBT test. This assay is performed by microscopically assessing the ability of individual cells to reduce NBT to purple formazan crystals following stimulation. Commonly there is no NBT reduction with most forms of CGD. In some of the variant forms, however, a high percentage of cells may contain some formazan, a finding indicative of a greatly diminished respiratory burst in most of the neutrophils. This test also permits detection of the carrier state in X-linked CGD when as few as 5 to 10 percent of the cells are NBT-negative.420
Most sophisticated procedures can identify the molecular defect. Cytochrome b content can be measured in extracts of detergent-disrupted neutrophils by a spectrophotometric assay.420 Once the diagnosis of CGD is made, the genotype can be determined. A mosaic population of oxidation that has positive and negative neutrophils in a male patient’s mother and sister strongly suggests X-linked CGD. Lack of a mosaic pattern among female relatives does not rule out the X-linked mode of inheritance because the defect can arise spontaneously. Prenatal diagnosis of CGD is established by analysis of DNA from amniocytes or chorionic villus samples.
Differential Diagnosis Leukocytes from patients with CGD have normal glucose-6-phosphate dehydrogenase (G6PD) activity. However, a few individuals with apparent CGD have been described who have neutrophils that lack or are almost lacking in G6PD activity.421,422 The erythrocytes of these patients also lack the enzyme, and the patients have chronic hemolysis. In the cases of severe neutrophil G6PD deficiency, an attenuated respiratory burst progressively decreases as a result of the depletion of intracellular NADPH, the primary substrate for the respiratory burst oxidase. CGD and G6PD deficiency can be distinguished from each other by the hemolytic anemia seen in the latter disorder and by the fact that erythrocyte G6PD activity is normal in CGD and markedly reduced in G6PD deficiency.401 A variety of studies indicate that the small GTPase Rac-2 plays an essential role in activity of the NADPH and the actin cytoskeleton in human neutrophils.383 A toddler has been described as presenting with a perirectal abscess at 5 weeks of age. This patient subsequently had necrosis of the periumbilical skin and fascia, and his surgical wounds did not heal properly. Functionally his neutrophils had multiple defective components; for example, adhesion to ligands for sLex, chemotaxis, release of primary azurophil granules upon stimulation with chemotactic peptide, and failure to undergo the respiratory burst using the same stimulus.423,424 Molecular analysis identified the asparagine for aspartic acid mutation at amino acid 57 of one allele of the Rac-2 gene.423,424 Mutant Rac-2 did not bind GTP and it inhibited and behaved as a dominant negative to impair Rac-2–mediated activation of the respiratory burst.424 Fortunately, the youngster was successfully transplanted with marrow from a HLA-identical older brother.424
Therapy, Course, and Prognosis Allogeneic hematopoietic stem cell transplantation is the only recognized curative treatment for CGD. Reduced intensity conditioning stem cell transplantation from HLA-matched donors performed in 56 patients with intractable infections and severe inflammation carried a 2-year overall survival of 96 percent.425 However, vigorous supportive care along with the use of recombinant IFN continues to be the foundation of treatment.376 Cultures must be obtained as soon as infection is suspected, as unusual organisms are commonly the source of infection and may grow promptly in vitro. Most abscesses require surgical drainage for therapeutic and diagnostic purposes, and prolonged use of antibiotics is often required. If fever occurs, it is advisable to obtain certain studies that aid in the management of septic episodes. These include roentgenograms of the chest and skeleton and a computed tomography (CT) scan of the liver because of the frequency of pneumonia, osteomyelitis, and liver abscesses.383 Arrangements should be made for prompt medical attention at the first signs of infection. With early intervention, many lesions can be managed by conservative medical means. For example, enlarging lymph nodes often regress when treated with local heat and orally administered antistaphylococcal antibiotics. It is particularly important to obtain a microbiologic diagnosis, and fine-needle aspiration may be helpful in this regard. In general, antibiotic therapy for the offending organisms is indicated and purulent masses should be drained. The cause of fever and prostration cannot always be established, and empiric treatment with broad-spectrum parenteral antibiotics is required. Often it is necessary to treat with antibiotics for a prolonged time until the initially elevated sedimentation rate approaches normal values. Aspergillus spp. infection requires treatment with amphotericin B or, in refractory cases, with granulocyte transfusions.376 Glucocorticoids also may be useful in the treatment of patients with antral and urethral obstruction. The risk of Aspergillus infection can be reduced by avoiding marijuana smoke and decaying plant material, such as mulch and hay, both of which contain numerous fungal spores.426 Long-term oral prophylaxis with trimethoprim-sulfamethoxazole (5 mg/kg per day of trimethoprim) is an accepted practice in the management of patients with CGD.376 Patients have prolonged infection-free periods, which result from the prevention of infections caused by S. aureus, without increasing the incidence of fungal infections. The use of itraconazole prophylactically has reduced the development of fungal infections.427,428
IFN-γ (50 mcg/m2, three times per week, subcutaneously) can reduce the number of serious bacterial and fungal infections.427,429 IFN-γ–enhanced neutrophil function in vitro has not been correlated with improvement in the activity of the neutrophil respiratory burst in patients totally lacking the ability to generate superoxide. On the other hand, its use increases the neutrophil expression of the high-affinity Fcγ receptor 1, as well as monocyte expression of FcγRI, FcγRII, FcγRIII, CD11/CD18, and HLA-DR.430 The IFN-γ protective effect in patients with CGD may involve improved microbial clearance, as suggested by the enhanced phagocytic activity by neutrophils of opsonized S. aureus. In rare, X-linked CGD patients able to generate some superoxide, IFN-γ programs granulocyte cells to increase their expression of cytochrome b, which results in normal superoxide generation.431 With the use of current prophylactic treatments, the mortality in CGD has been reduced to two patient deaths per year per 100 patients followed.376
CGD patients with mutations that result in 5 to 10 percent of normal-functioning amounts of NADPH have a mild phenotype and better clinical prognosis than do patients with complete absence of any NADPH-oxidase activity.432 Similarly, female carriers of X-linked CGD who have only 3 to 5 percent oxidase-normal neutrophils rarely get serious infections suggestive of the CGD clinical phenotype.433 Thus, even low levels or partial correction by gene therapy of CGD is likely to provide clinical benefits. In support of that hypothesis, mouse models of X-linked and p47phox-deficient CGD have been developed by gene targeting.434,435 Studies in the gp91phox- and the p47phox-deficient mouse models of CGD show that retrovirus-mediated gene-therapy-targeting of marrow progenitor cells ex vivo can result in the correction of defects in oxidant production in vivo in blood neutrophils after radiation conditioning and transplantation of marrow stem cells.436,437 Protection from infection challenge occurred even when the oxidase-corrected cells comprised less than 10 percent of circulating neutrophils. These promising results suggest that somatic gene therapy can be employed to correct defective phagocyte oxidase function in selected patients with CGD. In a phase I clinical trial, gene therapy for p47phox-deficiency CGD, five adult patients received intravenous infusions of autologous blood stem cells that were ex vivo transduced using a retrovirus encoding normal p47phox.438 Although conditioning therapy was not given prior to the stem cell infusion, functionally corrected neutrophils were detectable in blood for several months.339 In another study, long-term high-level clinical beneficial correction in ex vivo gene therapy of X-linked CGD occurred in two adult patients.439 Nonablative busulfan conditioning was used to augment gene therapy correction. There needs to be caution regarding the long-term stability and safety of gene therapy. For instance, there are concerns about gene insertion rendering patients vulnerable to developing an hematological malignancy.
The functional and immunochemical absence of the enzyme MPO from granules of neutrophils and monocytes, but not eosinophils, is inherited as an autosomal recessive trait, with a prevalence of 1:2000.440 MPO, an enzyme that catalyzes the production of HOCl in the phagosome. In MPO deficiency, the microbicidal activity of the neutrophils is reduced early after ingestion of microorganisms (see Table 66–2). However, normal microbicidal activity is observed in approximately 1 hour after a variety of organisms are ingested.440 Thus, the MPO-deficient neutrophil uses an MPO-independent system for killing bacteria that is slower than the MPO–H2O2–halide system, but that is eventually effective in eliminating bacteria. MPO-deficient neutrophils accumulate more H2O2 than do normal neutrophils; the higher peroxide concentration improves the bactericidal activity of the affected neutrophils. In contrast to the retardation of bactericidal activity, candidacidal activity in MPO-deficient neutrophils is absent.440 The most significant clinical manifestation in a few patients with diabetes mellitus and MPO deficiency has been severe infection with C. albicans. Because this is such a common disorder of phagocytes, it is important to note that the vast majority of patients with this genetic disorder have not been unusually susceptible to pyogenic infections and do not require therapy.
The complementary DNA encoding human MPO has been cloned and the gene structure, including promoter and regulatory elements, delineated.440 The gene consists of 12 exons and 11 introns and is located on the long arm of chromosome 17, and its expression is finely coordinated with expression of genes encoding other lysosomal proteins. Expression of genes for human neutrophil elastase and MPO is very similar; it is low in myeloblasts, peaks during the promyelocyte stage, and eventually drops to low levels in myelocytes. MPO is a symmetric molecule composed of four peptides, where each half consists of a heavy- and a light-chain heterodimer.440 Each heavy- and light-chain heterodimer starts as a single peptide that is cleaved during the posttranslational process to yield the heavy and light chains that form half of the mature molecules. The two halves of the molecule are associated by a disulfide linkage between heavy-subunit residues at their residue C319.
The primary translation product of the gene is a single-chain peptide of 80 kDa that undergoes cotranslational glycosylation at several asparagine residues, followed by a series of modifications of these oligosaccharides. The apopromyeloperoxidase exists for a prolonged time in the endoplasmic reticulum, where it associates reversibly with several endoplasmic reticulum–resident proteins known as molecular chaperones.440 Subsequent to heme insertion, the enzymatically active promyeloperoxidase undergoes proteolytic cleavage of the pro region. Then, in a prelysosomal compartment, the single peptide is cleaved into the heavy and light subunits, which remain linked. During final sorting within the azurophil lysosome compartment, there is dimerization of half-molecules to form the mature MPO.
Most patients with MPO deficiency have a missense mutation in the gene that results in replacement of arginine 569 with tryptophan.440 The mutation results in a precursor that associates with molecular chaperones, but does not incorporate heme, resulting in a maturational arrest during processing at the stage of an inactive enzymatic apopromyeloperoxidase. Other patients are heterozygotes with one allele bearing the common mutation and the other being normal, resulting in a partial deficiency.441 To date, four genotypes have been reported to cause inherited MPO deficiency, each of which results in missense mutations. In the genotype Y173C, a missense mutation results in replacement of a tyrosine at codon 173 with a cysteine residue resulting in the mutant precursor being retained in the endoplasmic reticulum by virtue of its prolonged interaction with the chaperone calnexin, and eventually undergoing degradation in a proteasome.440 In this way, the quality control system operating in the endoplasmic reticulum retrieves misfolded MPO precursors from the biosynthetic pathway and creates the biochemical phenotype of MPO deficiency. In another patient, a missense mutation resulted in an intact MPO molecule that acquired heme but failed to undergo proteolytic processing to a mature molecule.
Acquired disorders are associated with MPO deficiency. Reported states include lead intoxication, ceroid lipofuscinosis, myelodysplastic syndromes, and acute myelogenous leukemia.442 One-half of untreated patients with acute myelogenous leukemia and 20 percent of patients with CML may have MPO deficiency.442
Deficiencies of Glutathione Reductase and Glutathione Synthetase
Neutrophils contain enzymes capable of inactivating potentially damaging reduced oxygen byproducts. Disposal of superoxide anion is accomplished through superoxide dismutase, a soluble enzyme that converts superoxide to a H2O2. H2O2 is detoxified by catalase and by the glutathione peroxidase–glutathione reductase system, which converts H2O2 to water and oxygen.443 In addition to the soluble enzymes, cellular vitamin E serves as an antioxidant to prevent damage to the surface of activated neutrophils when releasing H2O2.443 Single cases of profound deficiencies in glutathione reductase444 and glutathione synthetase443 have been associated with impaired neutrophil bactericidal activity (see Table 66–2). Both deficiencies are associated with hemolysis under conditions of oxidative stress (Chap. 48). Glutathione synthetase deficiency also has been associated with intermittent neutropenia during times of mild infection. Vitamin E has been employed to ameliorate the hemolysis and improve neutrophil function in a patient with glutathione synthetase deficiency.445 Like patients with MPO-deficient neutrophils, the patients with glutathione reductase deficiency and glutathione synthetase deficiency are not unusually susceptible to bacterial infections.