PG Quie

Abstract

Patients with congenital disorders of the phagocytic system have served as "nature's own experiments" demonstrating the importance of specific functions of phagocytic cells for host defense against bacterial and fungal infections. Patients with Leukocyte Adherence Deficiency (LAD) stimulated research on surface receptors involved in margination and diapedesis of leukocytes from the circulation into tissues. Patients with Chronic Granulomatous Disease (CGD), who are unusually susceptible to serious infections from catalase positive bacteria and fungi, revealed the importance of a respiratory oxidative response of phagocytes. We have learned that products of oxidative metabolism are essential for efficient intracellular killing of microbes by neutrophils and macrophages. Patients with Job's Syndrome suffer recurrent infections related to dysfunction of neutrophils which may be secondary to cytokine dysregulation. These patients have taught us the importance of immunologic "discipline" for normal function. Patients described in this review with congenital disorders of the phagocytic system may be rare, but the lessons they provide are applicable to many more patients with acquired or transitory disorders of phagocytes. We continue to be amazed by this remarkably complex and essential host-defense system.

Introduction

The phagocytic system is an essential part of our host defenses necessary for life in this essentially microbial world. The phagocytic system consists of leukocytes (neutrophils, eosinophils, monocytes, and macrophages); opsonins (antibody, complement and lectins) and multiple cytokines necessary for regulation and control of the system. This will be a review of information about the role of cellular components of the phagocytic system, and one type of cell, the neutrophil (also called polymorphonuclear leukocyte (PMN) or granulocyte) will be emphasized. Children with congenitally acquired defective function of neutrophils are "nature's own experiments."¹ We owe these children a large debt of gratitude since most of the recent knowledge of phagocytic cell function has come from careful and persistent study of these patients and their cells.

Clinical presentation of patients with disorders of the phagocytic system include severe and recurrent infections which may involve any organ system; adenitis, pneumonia, liver abscess and osteomyelitis are common. Periodontitis and mouth ulcers may be chronic problems and sepsis, while unusual, may be life-threatening. Clinical signs and symptoms are similar in any disorder of the phagocytic system, therefore, laboratory evaluation of phagocytic cell numbers and function should be done in patients with recurrent infections that involve abscesses; are unusually severe; or respond inappropriately to antibiotics. A correct diagnosis is especially important in children with congenital disorders of the phagocytic system since gene therapy is on the horizon.² Furthermore, heterozygote carriers may be identified, which is useful for genetic counseling and prenatal diagnosis is possible.

Leukocyte Adhesion Deficiency

Leukocyte adhesion deficiency (LAD) is a disorder of the ability of neutrophils and other circulating leukocytes to adhere to particulate materials or endothelial cells. Leukocytes from patients with LAD are congenitally deficient in surface adhesion glycoproteins (CD 18 complexes).³ LAD is inherited in an autosomal recessive pattern and two clinical phenotypes have been identified.⁴ One phenotype has compete absence of CD 18 adhesion glycoproteins on leukocytes and these patients suffer severe life-threatening infections. Patients with diminished (3% to 20% of normal) glycoproteins on leukocytes have increased susceptibility to repeated infections, however they have less severe infections i.e. periodontitis, recurrent skin and mucous membrane infections with bacteria or fungi, and poor wound healing. LAD has been described in Chinese patients as well as in other nationalities.⁵

Newborn infants with LAD may have delayed separation of the umbilical cord (21 days or longer). Omphalitis with peritonitis and death from sepsis may occur in the neonatal period and older patients are frequently afflicted with otitis, sinusitis and pneumonia. Extreme neutrophilia (70,000/μl to 120,000/μl) is characteristic when infections occur, since neutrophils are released normally from the bone marrow in LAD patients in response to stimulation by C_3e interleukins and other inflammatory mediators.⁶ Adherence of leukocytes to blood vessel walls is defective, however neutrophils remain in the axial flow of blood vessels without migrating into tissues. When LAD is suspected clinically, the diagnosis may be confirmed by flow cytometric analysis of peripheral blood leukocytes using the monoclonal antibodies described in a recent report.⁵

The gene for CD-18 is on chromosome 21 and genes for alpha subunits are on chromosome 16. A variety of mutations in the gene for CD-18 have been described,⁷ including failure of the beta subunit to associate with alpha subunits in the endoplasmic reticulum, resulting in decreased expression of CD-18 CD-11 adhesion glycoprotein on neutrophil surfaces. Upregulation of these surface glycoproteins does not occur after stimulation of neutrophils with interferon gamma or tumor necrosis factor in some LAD patients, suggesting absent glycoprotein in specific (storage) granules as well as on neutrophil surfaces. Other LAD patients have defective m-RNA transcription for adhesion glycoproteins.⁸ Therapy with recombinant interferon gamma may increase m-RNA and produce effective levels of surface adhesion glycoproteins in LAD patients when mutations affect transcription.

The author and his colleagues have successfully managed an LAD patient with complete absence of neutrophil CD-18 CD-11 surface glycoprotein for 20 years. She had severe peritonitis as a newborn; recurrent sepsis and chronic progressive periodontitis as a child; and Candida lesions of mucous membranes as a young adult. Skin lesions heal extremely slowly. Each physical challenge has responded to antibiotics and other medical therapies administered earlier and more aggressively than usual. Her emotional and psychological resilience is remarkable. Other LAD patients have been successfully treated with bone marrow transplantation.⁹ An animal model LAD has recently been developed and CD-18 deficient lymphocytes have been corrected in vitro by retrovirus mediated gene transfer.¹⁰ Progress in the field of genetic engineering is extremely rapid and gene transfer may soon become practical therapy for LAD patients.

Chronic Granulomatous Disease

Chronic Granulomatous Disease (CGD) is a hereditary disorder of phagocytic cells.¹¹ Inheritance may be X-linked or autosomal recessive. All "professional" phagocytes i.e. neutrophils, eosinophils, monocytes and macrophages fail to respond to stimulation with a "respiratory burst" and reactive oxygen radicals, essential for efficient intracellular killing of certain bacteria and fungi, do not accumulate in phagocytic vacuoles. Phagocytes from CGD patients do not generate superoxide or other microbicidal radicals in response to particle phagocytosis or other stimulation.¹²

An abnormal oxidase, nicotinamide adenine dinucleatide phosphate oxidase (NADPH) is necessary for the one-electron reduction of molecular oxygen to superoxide which is rapidly dismutated to H₂O₂ and a variety of organic peroxides. NADPH oxidase is defective in phagocytic cells from all CGD patients. NADPH oxidase activation requires several protein components and therefore several genetic varieties of CGD have been discovered and described.¹³ Patients with CGD have been identified in all countries of the developed world and there appears to be no ethnic difference in prevalence of CGD. An incidence of 1 CGD patient per 1 million population in the United States and England is probably an underestimation since countries with accessible health care and accurate social systems, such as the Netherlands, Denmark and Sweden, have an incidence of 1 CGD patient per 160,000 to 250,000 individuals.¹⁴ The author's recent visit in Hong Kong and his experience with CGD in Minnesota suggests that CGD prevalence everywhere in the world is approximately 1 per 200,000 people.

When neutrophils and other phagocytic cells engulf microbes NADPH oxidase is activated, superoxide anions are formed which dismutate to hydrogen peroxide in the presence of chloride and become hypochlorous acid (HOd), which is cidal to most bacteria and fungi. The membrane bound part of NADPH oxidase consists of cytochrome b 558 with an alpha and beta subunit. The beta subunit is a glycosylated protein called gp9l phox and the alpha unit is protein called p22 phox. Both subunits must be present for activity. In addition to the membrane bound components, two and perhaps more proteins soluble in the cytoplasm are necessary for membrane associated NADPH oxidase activity.¹⁵ Activation involves translocation of cytoplasmic components of NADPH oxidase to the membrane. Several CGD patients have been reported lacking a cytosolic protein designated p47 phox and rare patients lacking another cytosolic protein p67.¹⁵ The gene for each of these NADPH oxidase components has been located.¹⁶ Gene locations are:

gp91 phox = Xp21.1
p47 phox = 7q11.23
p67 phox = 1q24
p22 phox = 16q24

Diagnosis of CGD depends upon measurement of the respiratory burst of stimulated neutrophils in the laboratory. The most simple diagnostic test available at most medical centers throughout the developed world is the nitroblue tetrazolium reduction test (NBT). Superoxide generated by NADPH oxidase activity when phagocytic cells from normal individuals are perturbated reduces the soluble colorless nitroblue tetrazolium to insoluble formazan which is deep blue in color. Since neutrophils and monocytes from CGD patients have defective NADPH oxidase response to stimulation, superoxide is not produced and NBT remains in solution and colorless; therefore CGD neutrophils do not contain blue formazan precipitate. Carrier mothers of boys with X-linked CGD are chimeric i.e. two populations of circulating neutrophils are observed on blood smears after stimulation in the presence of NBT. CGD carriers have a consistent percentage of normal NBT reducing neutrophils, however, different carrier mothers have percentages of normal cells ranging from 5% to 80%. Extreme Lyonization i.e. inactivation of either the normal or abnormal X-chromosome is believed to occur early in embryonic development and "the progeny of those cells continue to express only the set of genes remaining active after that event."¹³ It has been determined that inactivation occurs when eight embryonic hematopoietic cells are present in embryogenesis.¹⁷ The two populations of peripheral blood neutrophils in CGD carriers may also be determined by flow cytometric assays with dihydroshodamine. Indeed this method allows diagnosis of CGD in small amounts of whole blood.¹⁸ Cytochrome C reduction and chemiluminescence are alternative ways for determining superoxide production by neutrophils and therefore diagnosis of CGD and the carrier state.¹⁹ Monoclonal antibodies are available so that specific genetic defects can be identified by immunoblots directed against NADPH oxidase components gp91 phox, p22 phox, p47 phox and p67 phox. Both the gp9l phox and p22 phox must be present for a band to appear in gel electrophoresis, therefore determining carrier state of the mother is important for separating X-linked CGD (missing gp9l phox) from autosomal recessive CGD missing p47 phox - p67 phox or p22 phox. Prenatal diagnosis is possible by analyzing fetal DNA obtained by chorionic villus biopsy or using specific monoclonal antibodies against the four known gene product abnormalities associated with CGD. Identification of polymorphism within the gp9l phox gene may also be useful for prenatal diagnosis of CGD.²⁰

The gene for gp91 phox is on the X chromosome and it was the first human gene cloned on the basis of chromosomal location.²¹ All CGD patients identified to date have a molecular defect in one of four protein components of NADPH oxidase.¹⁹ Most of the patients are male with X-linked inheritance (55 - 75%) and are missing or have defective gp9l phox. The others may be male or female, with autosomal recessive inheritance and absence of one of the cytosolic proteins p47 phox or p67 phox. CGD patients with mutations in the genes for the 22K protein of the membrane bound glycoproteins have also been identified.²² Some CGD patients have low but detectable levels of cytochrome b558 and with NADPH oxidase activity between 5% and 25% of normal. These patients have a more benign clinical course and are referred to in the literature as CGD variants. More than 40 different mutations have been found in the genes for both the gp91 phox gene and the p22 phox gene. These include point mutations leading to missense with in-frame exchange of amino acid. Nonsense leading to premature stop codon as well as deletions and insertions.²³

Children with X-linked CGD often present to physicians during the first two years of life with unusual infections. Examples of these infectious problems are pneumonia which responds slowly to appropriate antibiotics and abscess lesions of lymph nodes, liver or the perirectal area. CGD patients with autosomal recessive inheritance have fewer and less severe infections.¹⁴ Organ systems most heavily exposed to large numbers of bacteria and fungi are major sites of infection in CGD patients including lungs, skin, GI tract, liver and lymph nodes which drain these organs.²⁴ Sepsis is relatively unusual, although osteomyelitis from hematogenous distribution is not uncommon. CGD patients have a typical response to infection with fever, leukocytosis and appropriate inflammatory response with elevated sedimentation rate and other acute phase reactants. Incomplete resolution of abscesses or areas of inflammation results in granulomatous lesions characterized by giant cells with lipid material, fibroblasts and eosinophils. These granulomas contribute to morbidity by obstructing the GI or GU tract. Healing of surgical wounds and resolution of GI or GU obstruction is often delayed.²⁵

The bacterial and fungal species which infect CGD patients are invariably catalase positive. Catalase negative organisms are killed normally by CGD neutrophils presumably because bacterial metabolism contributes sufficient hydrogen peroxide and enough hypochlorous acid is formed within phagocytic vacuoles to kill ingested organisms. The microbial agents frequently associated with severe disease in CGD patients are:

Staphylococcus aureus
Serratia marscesens
Aspergillus fumigatus
Pseudomonas cepacia
Klebsiella species
Nocardia
Mycobacterium tuberculosis

Several other opportunistic microbial species reported to be associated with disorders of phagocytic cells are:¹⁹

Legionella species
Chromobacterium violaceum
Candida albicans
Franciella philomiragia
Hansenula polymorphia
Paecilomyces varioti
Pseudallerscheria boydii
Pneumocystis carinii

Many CGD patients have onset of pulmonary symptoms after exposure to environments potentially saturated with Aspergillus spores including grain storage areas, barns with musty hay, moldy leaves and damp basements. Microbes listed above may be present in high concentrations in contaminated fresh as well as salt water. Swimming for CGD patients must be restricted to water that is chlorinated.

As noted above, the microbial species associated with severe disease in CGD patients include Gram-positive and Gram-negative bacteria and fungi. A common denominator of these species is catalase production. Catalase negative species are killed normally by CGD neutrophils.

Prophylaxis is difficult because of different antibiotic sensitivities. A universally recommended prophylactic treatment is daily trimethoprim- sulfamethoxazole.²⁶ However, patient sensitivity to sulfamethoxazole in this compound limits its usefulness. Prophylactic trimethoprim is recommended for patients sensitive to sulfa and dicloxacillin may be added for prophylaxis of Gram-positive bacteria. Older patients, i.e. teenagers and beyond, may be treated with one of the quinolones, usually ciprofloxacin.

Aspergillus is a dreaded fungal agent associated with CGD. Until recently, prophylaxis was not possible and treatment of pulmonary infections and osteomyelitis required months of Amphotericin B. More recently, however, itraconazole has been approved for treatment of Aspergillus infection. A 1994 prospective study of 32 CGD patients in France given itraconazole suggests that itraconazole may also be effective as prophylaxis.²⁷

The cytokine, human recombinant interferon gamma (IFN-γ) is recommended as prophylactic therapy for CGD patients. An international study of 132 patients with CGD showed that IFN-γ reduced the number of serious infections and number of days of hospitalization when infections occurred.^28-29 IFN-γ is administered at a dose of 50 μg/m² three times weekly as a subcutaneous injection. Except for flu-like symptoms easily controlled with acetaminophen, IFN-γ has minimal side effects.

While infections continue to occur in his CGD patients receiving IFN-γ, the author is impressed that the interval between infections is longer and response to appropriate antimicrobial treatment is more normal in IFN-γ treated CGD patients and the rate of infections has not increased in patients given IFN-γ for several years.

The mechanism of action of IFN-γ is unknown. A minority of CGD patients with a measurable amount of NADPH oxidase activity (so-called CGD variants) have a demonstrated increase in cytochrome b558 gene expression and NADPH oxidase activity after IFN-γ treatment.³⁰ However, no difference was observed in NADPH oxidase response in neutrophils from most CGD patients treated with IFN-γ during the international trial. A young Swedish investigator reported recently that CGD neutrophils treated with IFN-γ have increased nitric acid production and increased opsonin receptor activity.¹⁴

Aggressive, early treatment with intravenous, high-dose broad spectrum antibiotics are essential when infections do occur in CGD patients despite IFN-γ and prophylactic antibiotics. Although antibiotics such as rifampin that reach high levels inside neutrophils have a theoretical advantage, it is the author's experience that bactericidal antibiotics chosen on the basis of in vitro sensitivities are superior. Surgery is frequently necessary for resolution of pyogenic abscesses. Transfusion of leukocytes may also be useful in selected life-threatening conditions such as liver, brain or lung abscesses that persist in spite of aggressive antibiotic therapy and attempts at surgical drainage. A frequent complication of infections involving the GI or the GU tract is granulomatous obstruction; indeed obstructive lesions may be the first clinical manifestation of CGD. Often steroids are necessary (together with broad spectrum antibiotics) and accelerate relief of pain and restoration of more normal function.¹⁹ Bone marrow transplantation has been attempted in several CGD patients and successes have been reported.³¹ Even more promising, however, is the realistic expectation that gene therapy may soon be applied to correct NADPH oxidase system in CGD hematopoietic progenitor cells. Patients with CGD are ideal candidates for gene therapy since hematopoietic progenitor cells are easily available, the involved genes have been cloned, and retroviral vectors have been transfected with the gene for both X-linked and autosomal recessive forms of CGD.^32,33

Jobs' Syndrome (Hyperimmunoglobulin E Syndrome)

Jobs' Syndrome is a familial disease and both males and females are affected, suggesting autosomal dominant inheritance. This clinical syndrome may be primarily related to immunoglobulin or cytokine dysregulation and disorder of the phagocytic system may be secondary. Patients with Jobs' Syndrome have elevated levels of serum IgE (over 2,000 IU/ml). Dermatologic lesions begin with eczema-like lesions heavily colonized with Staphylococcus aureus, which become pustular and, in older patients, are lichenified. When abscesses occur, they are often "cold" i.e. little pain or redness or surrounding cellulitis.³⁴ S. aureus is often cultured from abscesses and although neutrophils are identified in abscess material, smaller than usual numbers are present. Chronic otitis media and mastoiditis are common and peritonsillar abscesses may obstruct the airway.³⁵

Coarse facial features consisting of a broad nasal bridge, prominent nose and irregularly proportioned maxillary and jaw development are progressive as Jobs' Syndrome patients become older. The characteristic growth retardation and facial features suggest that osteoclasts may be abnormally regulated in this disease. Osteoporosis is associated with frequent fractures and chronic arthritis of wrists and interphalangeal joints. Recurrent severe adenitis and pneumonia occurs frequently in Jobs' Syndrome patients and unless pulmonary infections are treated promptly and aggressively, pneumatoceles and empyema develop. Prophylactic treatment with antistaphlococcal antibiotics is recommended.

Elevated levels of serum IgE especially when noted during early months of life and in patients with several abscess lesions suggest the diagnosis of Jobs' Syndrome. Serum levels are typically 20,000 IU or higher in older children and adults and eosinophilia is another laboratory characteristic of Jobs' Syndrome. Percentages of eosinophils may reach 80%, however the number of circulating neutrophils are usually lower during the leukocytosis of acute infection. A more specific diagnostic laboratory test is measurement of anti-Staphylococcus aureus IgE using a RAST test. Jobs' Syndrome patients have higher levels of anti-staphylococcus IgE antibodies than patients with atopy.³⁶ Immunologic abnormalities in Jobs' Syndrome include the T-cell system, B-cell system and the phagocytic system. Patients have decreased numbers of CD8 "suppressor cells," therefore immunoglobulin E producing B-cells are not inhibited. Mononuclear cells from patients with Jobs' Syndrome produce an inhibitor of neutrophil chemotaxis continuously, which is normally produced after stimulation, suggesting that cytokine dysregulation may be the basis for immunologic and phagocytic functional disorders in Jobs' (hyperimmunoglobulin E) Syndrome.³⁷ A recent report that mononuclear cells from Jobs' Syndrome patients have defective production of interferon γ, a physiologic inhibitor of IgE production, suggests that treatment with recombinant human IFN-γ may be beneficial.³⁸ Tumor surveillance may also be defective since Hodgkin's Disease and other lymphomas have been diagnosed in several Jobs' Syndrome patients.³⁹

As in most immunodeficient patients, early aggressive diagnosis and treatment of infections is a key requirement to prevent life-threatening complications such as pneumatoceles and obstructive lung abscesses. The eczematoid dermatitis that is characteristic in Jobs' Syndrome responds to topical steroids, but frequently requires systemic antistaphlococcal antibiotics as well. Frequent Candida lesions of mucous membranes are distressing, but are fairly easily controlled with antifungals. Several adjuvant therapies such as levamisole, vitamin C, cimetidine and intravenous gamma globulin have been tried, but have not proven to be beneficial. Daily attention to skin care, prophylactic antibiotics and aggressive treatment with antibiotics when infections occur results in a reasonable quality of life for most Jobs' Syndrome patients.

Kostmann's Syndrome

Patients with Kostmann's Syndrome have a congenital absence of circulating neutrophils due to arrest of myelopoiesis at the promyelocyte stage in the bone marrow. Severe infections of the upper and lower respiratory tract begin during the first 6 months of life and until recently, less than 50% survived until their first birthday. The recent clinical application of recombinant human granulocyte colony stimulating factor for patients with congenital neutropenia (Kostmann's Syndrome) has made a dramatic difference in prognosis.⁴⁰ A basic defect of myeloid cells in this syndrome is defective ability to transduce maturation signals. Fortunately, capacity for normal proliferation and differentiation to mature cells is present and infants with Kostmann's Syndrome usually respond to granulocyte colony-stimulating factor with substantial increase in numbers of circulating neutrophils. When infections occur in spite of growth factor administration, treatment with intravenous antibiotic needs to be aggressive and broad spectrum, because endogenous flora are often causative and polymicrobial infections are common.

Cyclic Neutropenia

Cyclic Neutropenia is a congenital disorder or neutrophil numbers and is usually inherited as autosomal dominant with variable expression.⁴¹ Patients with Cyclic Neutropenia have regular cycles (usually every 21 days). Therefore, numbers of circulating neutrophils are dangerously low i.e. less than 200 cells per mm³ for 2-8 days and with gradual return to normal levels approximately once each month. The cycle of monocytes and eosinophils may be a "mirror image" of the neutrophil cycle so that total number of circulating leukocytes is near normal. During the days of severe neutropenia, mouth ulcers, sore throat and painful lymph node enlargements are often present. Infectious complications have their onset during the neutrophil nadir, however when medical attention is received, i.e. several days after onset of the illness, neutrophil numbers may be near normal. Neutrophil numbers need to be documented three times per week for six weeks to diagnose cyclic neutropenia. Myeloid progenitor cells are present in high concentrations in the bone marrow of cyclic neutropenia patients but are delayed in differentiation and maturation in response to physiologic stimulators. Treatment with pharmacologic concentrations of recombinant human granulocyte colony-stimulating factor results in increased number of neutrophils throughout the cycle and this improvement in host defenses greatly reduces frequency and severity of infections.⁴¹

Chédiak-Higashi Syndrome

The Chédiak-Higashi Syndrome is an extremely rare disorder estimated to have a prevalence of one per ten million persons. The syndrome is characterized clinically by frequent, relatively mild infections, partial albinism, hepatosplenomegaly, and in some patients a progressive neuropathy.⁴² All granule containing cells including neutrophils and monocytes, even platelets and lymphocytes contain giant cytoplasmic inclusions. Diagnosis of Chédiak-Higashi Syndrome is made by Wright's stain of peripheral blood.

The inclusions in phagocytic cells are accumulations of liposomes believed a consequence of disordered assembly of microtubules and abnormal tubulin tryosinolation.⁴³ The Chédiak-Higashi Syndrome may be familial, however the genetic inheritance has not been determined. Phagocytic cell chemotaxis is depressed as a consequence of abnormal "giant" cytoplasmic inclusions. Neutrophil and monocyte bactericidal activity is depressed because the "giant" cytoplasmic inclusions remain intact during phagocytosis and do not discharge liposomal enzymes into phagocytic vacuoles. Intracellular killing of Staphylococcus aureus and Candida albicans is compromised.⁴⁴ Treatment of Chédiak-Higashi Syndrome patients is similar to that described for patients with other disorders of phagocytic cells i.e. prompt and aggressive diagnosis and treatment when infections occur. Bone marrow transplantation has recently been successfully utilized for treatment of Chédiak-Higashi Syndrome.⁴⁵

Conclusion

Patients with congenital disorders of the phagocytic system are few in number, but their contributions to our knowledge of normal host defense against bacterial and fungal diseases is enormous. Our gratitude is expressed by continuing investigation of cells and application of this newly acquired knowledge to provide new therapies for these patients and for much larger numbers of patients with secondary or acquired disorders of phagocytic cells. It is expected that genetic studies of patients with abnormalities of phagocytic cell function will continue to reveal "nature's own secrets' and this knowledge will lead to therapies that will replace defective hematopoietic cells with "corrected" cells capable of differentiation and maturation to mature neutrophils and monocytes.

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