Functional Disorders of Granulocytes

C. Wayne Smith

M. Michele Mariscalco

Mobile blood granulocytes and monocytes and fixed phagocytic cells function as a first-line defense against invasion by bacterial or fungal microorganisms. Impaired granulocyte production, as well as the functional abnormalities of granulocytes or other professional phagocytes, may significantly compromise host defense, thus increasing susceptibility to infection. Early animal studies demonstrated a critical 2- to 4-hour period after cutaneous invasion by pathogenic bacteria during which phagocytes must localize at a site of invasion to prevent or suppress the process of infection. Recurrent bacterial or fungal infections of the skin or mucous membranes are prominent in patients with quantitative deficiencies of blood granulocytes and in patients with functional deficits of granulocytic cells.

Two broad categories of functional disorders have been delineated: those typified by impaired motility, recruitment, or localization of granulocytes at or to sites of infection, and those resulting from the defective ingestion or intracellular killing of microorganisms by granulocytes and other phagocytic cells. In this latter group of patients, granulocytes accumulate normally in inflamed tissues but are unable to eradicate invading microorganisms. Laboratory studies of representative patients of both categories can be used to define abnormalities of one
or more cellular functions (e.g., directed migration or chemotaxis, adhesion, ingestion, degranulation, oxidative intracellular killing, or all) in vitro. In selected disorders, molecular deficits have been defined, which allows important new approaches to the diagnosis or clinical management of disease.

DISORDERS OF ADHERENCE AND MOTILITY

Leukocyte Adhesion Deficiency Type I

Leukocyte adhesion deficiency type I (LAD I) is an autosomal recessive trait characterized by recurrent bacterial infections, impaired pus formation and wound healing, and a spectrum of functional abnormalities in granulocytes, monocytes, and lymphoid cells (Box 430.1).

Clinical Manifestations and Complications

The clinical hallmarks of this disease are recurrent, necrotic, and indolent infections of soft tissues, primarily involving skin, mucous membranes, and the intestinal tract. Superficial infections of body surfaces may invade locally or systemically. Typical small, erythematous, nonpustular skin lesions often progress to large, well-demarcated, ulcerative craters (or pyoderma gangrenosa), which heal slowly or with dysplastic eschars. Staphylococcal or gram-negative enteric bacterial organisms may be cultured from such lesions for several weeks, despite antimicrobial therapy. Septicemia progressing from omphalitis associated with delayed umbilical cord severance has been observed in several families. Perirectal abscess or cellulitis leading to peritonitis or septicemia has been reported in multiple patients, and facial or deep neck cellulitis has been observed to progress from ulcerative mucous membrane lesions of the oral cavity. Recurrent invasive candidal esophagitis, erosive gastritis, acute appendicitis, and necrotizing enterocolitis have been reported in multiple patients. Recurrent otitis media occurs commonly, and progression to mastoiditis and facial nerve paralysis has been reported. Other common respiratory infections include severe bacterial (pseudomonal) laryngotracheitis, recurrent pneumonitis, and sinusitis. Severe gingivitis or periodontitis is a major feature among all patients who survive infancy. Acute gingivitis has appeared in all cases upon the eruption of the primary dentition. Subsequently, these patients develop characteristic features of progressive generalized prepubescent periodontitis, including gingival proliferation, defective recession, mobility, pathologic migration, and advanced alveolar bone loss associated with periodontal pocket formation and the partial or total loss of both the deciduous and permanent dentition.

BOX 430.1 Leukocyte Adhesion Defect

The molecular basis of LAD has been found to involve a family of structurally and functionally related glycoproteins on the surface of myeloid cells. These glycoproteins occur as noncovalently associated alpha and beta subunits with alpha₁-beta₁ stoichiometry. They share an identical beta subunit (Mr = 95,000) and are distinguished by distinct alpha subunits designated Mac-1-alpha (aM) (Mr = 165,000), LFA-1-alpha (αL) (Mr = 177,000), p150,95-alpha (αX) (Mr = 150,000), and alpha-D (Mr = 160,000). The World Health Organization designation for these glycoproteins is CD18 for the beta subunit, CD11a for the alpha-L subunit, CD11b for the alpha-M subunit, CD11c for the alpha-X subunit, and CD11d for the alpha-D subunit. The entire complex is designated CD11/CD18. Biosynthetic studies show that these patients possess heterogeneous abnormalities of the beta (CD18) but not alpha subunits of the CD11/CD18 glycoproteins. These findings suggested distinct mutations in the CD18 gene, which has been mapped to chromosome 21. Such mutations have been defined in several patients and include point mutations, deletions, or insertions, as identified by the nucleotide sequence analysis of patient or heterozygote CD18 cDNA or genomic DNA. Although most LAD phenotypes are not defined at the nucleotide level, it is probable that all include mutations of the CD18 gene and that CD18 mutants vary in their ability to complex with the different alpha subunits, thus determining the relative deficiency of the alpha-beta (αβ) protein complexes expressed on cell surfaces.

The recurrent infections observed in affected patients appear to reflect a profound impairment of leukocyte mobilization into extravascular inflammatory sites. Skin windows and biopsy samples of infected tissues demonstrate inflammatory infiltrates totally devoid of neutrophils. The histopathologic feature is particularly striking, because marked peripheral blood leukocytosis (9 to 20 times normal values) is a constant feature of this disorder. Transfusions of leukocytes result in the appearance of donor neutrophils and monocytes in skin windows and skin chambers. The dysregulated healing of traumatic or surgical wounds observed in several patients represents a clinical feature not generally observed in patients with neutropenia or dysfunctional neutrophils. Unusual, paper-thin or dysplastic cutaneous scars have been found in some patients and, in one patient with a unique CD18 mutation, hypertrophic scars occurred.

The severity of infectious complications among LAD patients appears to be related directly to the degree of glycoprotein deficiency. Severely affected patients have essentially undetectable expression of all three alpha–beta (αβ) complexes in their neutrophils. Moderately deficient patients express 2% to 25% of all three αβ complexes. Patients with severe deficiency have either died in infancy or have demonstrated a susceptibility to severe, life-threatening systemic infections (peritonitis, septicemia, pneumonitis, aseptic meningitis). In contrast, among the patients expressing some functional CD11/CD18 integrins, life-threatening infections have been observed infrequently despite a relatively prolonged survival (greater than 50 years). In some moderately affected patients, skin lesions may disappear after the first few years of life, recurring only with occasional infections. Severe gingivitis always is observed in these patients and may be the presenting symptom. Delayed umbilical cord separation occurs more frequently in patients with the severe phenotype, but it is not universally found.

Therapy

Suggested therapeutic guidelines for LAD are based on limited clinical experience. Although infectious complications generally are observed on body surfaces, life-threatening systemic infections may occur at any time, especially in individuals who fail to express any CD18 integrins. Superficial inflammatory lesions must be managed aggressively, with local care and antibiotic therapy. This is of special importance, because inflammatory signs may be minimal before the development of septicemic episodes. Moreover, the impaired healing of superficial
wounds appears to allow indolent colonization and subsequent reinfection. Early use of empiric combination therapy with a staphylocidal agent and an aminoglycoside is justified in acutely ill or febrile patients, in the absence of localizing findings. The use of prophylactic antibiotics may be advised. Limited clinical experience suggests that the number of systemic infections in patients with LAD is considerably diminished through the use of prophylactic regimens. Leukocyte transfusions have been used successfully in several patients with LAD; enhancement of inflammatory functions and clinical resolution of even life-threatening infections have been achieved in a clinical setting in which systemic antibiotic or surgical interventions were ineffective.

Bone marrow transplantation (BMT) with successful engraftment and apparent clinical recovery from disease has been achieved in several patients. Because of the inherent risks and expense, this approach should be considered only for patients with the severe phenotype of disease. Among patients undergoing transplantation, recipients of HLA-identical as well as HLA-mismatched (e.g., haplotype match) bone marrow have shown successful engraftment. In some cases, mixed but stable chimerism between donor and recipient cells is apparent 3 to 5 years after transplantation. In these cases, normal or slightly diminished blood granulocyte function is observed in vitro, and the patients demonstrate no clinical complications.

The identification in LAD patients having CD18 mutations raises the possibility that the introduction of a normal CD18 gene into hematopoietic cells could cure this disease, especially in light of the encouraging results of BMT. Some reports document the successful transfection or retroviral-mediated infection of LAD cells or cell lines with a normal CD18 cDNA, resulting in normal CD18 protein expression and normal cell adherence properties. Thus, the stage is set for future attempts at somatic cell gene therapy in LAD.

Leukocyte Adhesive Deficiency Type II

Leukocyte adhesive deficiency type II (Rambon-Hasharon syndrome, LAD II) is a rare condition characterized by the defective formation of ligands for the selectin adhesion molecule family. The syndrome is complex in that developmental abnormalities, mental retardation, and recurrent infections occur. It has been described in three kindreds, two of Middle Eastern descent and one of Brazilian origin. Each of the kindreds has distinct mutations in the GDP-fucose transporter and, functionally, a marked reduction in GDP-fucose transport into the Golgi apparatus. The distinctive clinical features of these patients include craniofacial dysmorphism, neurologic deficits (central hypotonia, seizures, developmental delay), recurrent respiratory infections, and constant peripheral blood leukocytosis and neutrophilia. An autosomal recessive genetic basis for this syndrome in these patients is supported by the finding that all lack the erythrocyte H antigen and manifest the Bombay (hh) phenotype that, in turn, is caused by a homozygosity for a rare recessive (h) allele. Individuals with the Bombay phenotype lack the H antigen, an intermediate in the production of erythrocytic A and B antigens, but do not share any other clinical features of LAD II.

The finding of markedly elevated neutrophil counts and recurrent respiratory infections in these patients prompted studies of leukocyte functions. These studies revealed diminished neutrophil emigration into skin windows and significant defects of random and directed migration and diminished homotypic aggregation of neutrophils in vitro. Studies of phagocytosis and intracellular bacterial killing, as well as lymphocyte proliferation and natural killer (NK) cell function, showed no abnormalities. In contrast to those of patients with LAD I, the leukocytes of these patients demonstrated normal levels of surface CD11/CD18.

LAD II patients have a generalized fucosylation defect that affects the alpha-1,2-, alpha-1,3-, alpha-1,4- and alpha-α1,6-linkages of fucose in glycoconjugates. These apparently result from mutations in a GDP-fucose transporter in the Golgi membrane. Two specific mutations have been discovered: the replacement of arginine 147 by cysteine (R147C) functionally inactivates the transporter and represents the defect in one kindred, and replacement of threonine 308 by arginine (T308R) results in the defective transporter in another kindred.

Dietary supplementation of fucose has been shown to reverse effectively the deficit of selectin ligands in two of the kindreds, with resulting improvement is some of the manifestations of the syndrome (e.g., normalization of the blood neutrophil count). It remains unclear whether oral fucose will significantly alter the developmental aspects of the syndrome, and it appears that one of the defined mutations is unresponsive to fucose supplementation. In addition, in one of the patients, a complication of inducing fucosylation was the appearance of neoantigens on the surface of leukocytes and erythrocytes. This patient exhibited an autoimmune neutropenia.

Leukocyte Adhesion Deficiency Type III

Leukocyte adhesion deficiency type III is a category of rare syndromes exhibiting the normal expression of the leukocyte adhesion molecules but abnormal functioning (previously referred to as LAD I variants). Several patients have been described with reduced adhesive functions in leukocyte integrins of the beta₁, beta₂, and beta₃ families. One patient exhibited a disorder in the multistep process of leukocyte adhesion to endothelial cells; this disorder was associated with functional defects in multiple leukocyte integrins, recurrent infections, marked leukocytosis, and a bleeding tendency. This syndrome was associated with an impaired ability of neutrophil and lymphocyte beta₁ and beta₂ integrins to generate high-avidity adhesion to endothelial ligands and arrest cells on vascular endothelium in response to endothelial chemoattractant signals. In this patient, a defect occurred in the regulation of the small Ras-related GTPase, Rap1, activation that resulted in an inability of ligand-occupied integrins to generate high-avidity binding to ligands under shear flow. Another patient exhibited clinical features of both Glanzmann thrombasthenia and LAD I. The patient had normal expression of beta₁, beta₂, and beta₃ integrins, but all were dysfunctional. The key findings were that signaling pathways leading to integrin activation were defective, and this defect was associated with abnormal integrin clustering. Although this group of patients likely represents several distinct molecular deficits, they share the characteristic of a dysfunctional family or families of leukocyte integrins that variably affect all leukocytes, sometimes including platelets. The fact that they express normal levels of integrins distinguishes them from LAD I patients.

Specific Granule Deficiency

The first example of a primary deficiency of neutrophil-specific granules was recognized in 1972. Other cases have subsequently been reported by several laboratories. One patient appears to have had an acquired deficiency (associated with a myeloproliferative syndrome), whereas all others appear to have genetically determined disease. Each demonstrated susceptibility to recurrent and severe infections of the skin, mucous membranes, and lung, most commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa, other enteric pathogens, and Candida albicans. Infections may progress from superficial
sites; otitis media with associated mastoiditis was reported in one patient, and lung abscess formation caused by S. aureus followed the onset of pneumonia in another individual. The occurrence of necrotic oral lesions caused by Escherichia coli and species of Pseudomonas and Klebsiella were reported in another individual, but the severe neutropenia recognized in that patient may have accounted for the development of these mucous membrane lesions. Another patient with severe scalp infections caused by Proteus mirabilis and S. aureus required prolonged intravenous antibiotic therapy in addition to surgical débridement. Detailed descriptions of the histopathology of infected tissues in all patients are not reported, but skin window studies have demonstrated diminished pus formation in the tissues of some individuals who were not neutropenic.

Clinical Manifestations and Complications

Neutrophils from each patient studied have demonstrated morphologic abnormalities, including a severe or total deficiency of specific granules and a variety of nuclear abnormalities, such as bilobed or multilobed nuclei or nuclear blebs, clefts, or pockets. Diminished or absent neutrophil lactoferrin content has been confirmed in only three cases, and the membrane marker alkaline phosphatase has been shown to be diminished or absent in the neutrophils of all but one reported case. Total cellular content and release of the secondary granule markers (lactoferrin, B12 transport protein, cytochrome b, and lysozyme) have been shown to be diminished when assessed in selected patients, although the levels of primary granule constituents (myeloperoxidase, beta-glucuronidase) generally are normal. Among recognized cases, somewhat heterogeneous abnormalities in cellular functions have been observed. Chemotaxis and intracellular microbial activity represent the most consistently reported functional deficits.

The deficiency of specific granules is suggested by a history of recurrent cutaneous, subcutaneous, mucous membrane, or pulmonary infections caused by S. aureus, virulent gram-negative enteric bacteria, or species of Candida. Findings of abnormal morphology and abnormally weak cytochemical reactions for alkaline phosphatase are highly suggestive of this disorder. Cytochemical and ultrastructural studies to confirm the diminished numbers or abnormal morphology of specific granules and their specific constituents establishes a diagnosis.

Sequence analysis of DNA from a patient with secondary granule deficiency revealed a five base-pair deletion in the second exon of the transcription factor CCAAT/enhancer binding protein–ε (C/EBPε) locus. A second patient had a one base-pair insertion, and the predicted frame shifts resulted in truncations of the 32-kD major C/EBPε isoform, with loss of the dimerization domain, DNA binding region, and transcriptional activity. The phenotypic and functional defects of neutrophils from C/EBPε-deficient mice closely parallel those of the human syndrome of specific granule deficiency. The multiple functional defects observed in early neutrophil progenitor cells are apparently the consequence of C/EBPε deficiency. Specific granule deficiency is thus a defect in myelopoiesis, because C/EBPe is required for the promyelocyte–myelocyte transition in myeloid differentiation.

Functional Abnormalities in Neonatal Neutrophils

Because specific immunity is limited severely in the immediate postpartum period, the inflammatory functions of phagocytic cells are especially important for host defense against microbial invasion. Both quantitative and qualitative abnormalities of phagocytic cells contribute to enhanced susceptibility of neonates to infections. Neutropenia is commonly observed in systemically infected neonates, and studies in neonatal animals indicate that the exhaustion of a limited reserve pool of bone marrow granulocytes contributes to a depletion of circulating or marginating pools when tissue demand is increased. Among the most consistently observed functional abnormalities thought to contribute to impaired inflammation in neonates are those related to the motility of leukocytes. As shown with skin windows, the inflammatory responses in newborns differ from those in older children and adults in two respects: The shift from the early granulocyte predominance to a predominance of mononuclear cells is slower and less pronounced, and a marked eosinophilia is observed in some infants aged 2 to 21 days. Strikingly diminished leukocyte mobilization in neonatal rats inoculated intraperitoneally with bacteria or chemotactic agents has been demonstrated.

Neonatal neutrophils exhibit impaired chemotactic responses to numerous chemotactic factors, including those released by replicating S. aureus and E. coli and those generated in plasma by antigen–antibody complexes (e.g., C5a). Visual assays demonstrate that neonatal cells not only have depressed migration but also are impaired significantly in their ability to orient toward a gradient of chemotactic factors. Depressed chemotaxis has been found in healthy neonates aged 1 to 5 days. In addition, diminished generation of chemotactic activity (chemotaxigenesis) by virulent type III group B streptococci occurs in neonatal sera, an abnormality directly related to diminished levels of both type-specific anticapsular antibody and serum complement activity. Thus, the impaired generation of chemotactic stimuli, as well as abnormal cellular responses, appear to account for the diminished inflammatory responses observed in even healthy term neonates.

The basis for abnormal neonatal granulocytic migratory functions is not defined fully but appears to involve defects of stimulated cell adhesion. The induction of CD11b/CD18 (Mac-1) surface expression by chemotactic agonists is diminished, and basal levels of the L-selectin adherence protein are markedly diminished on cord blood or neonatal granulocytes. The importance of these observations is suggested by findings of diminished transendothelial migration by neonatal neutrophils in vitro. This defect reflects impaired Mac-1–adhesive interactions with the endothelial ligand intracellular adhesion molecule 1 (ICAM-1) and deficits of L-selectin interactions with another endothelial adherence molecule, E-selectin. Studies of inflammation in neonatal animal models support a role for both neutrophil adhesion determinants in diminished inflammation.

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