Drug-induced agranulocytosis: in vitro evidence for immune suppression of granulopoiesis and a cross-reacting lymphocyte antibody.

Two patients with agranulocytosis associated with diphenylhydantoin (DPH) therapy and clinical data suggesting suppression of granulopoiesis were investigated using in vitro culture techniques for committed granulocyte/macrophage precursors. Addition of DPH to cultures containing the patients' sera resulted in significant suppression of colony growth. Extensive studies on the acute serum from one patient revealed the drug-dependent inhibitory activity to be nondialyzable, resistant to chloroform extraction, heat stable, active in the presence of heat-inactivated fetal bovine serum, active against autologous as well as allogeneic cells, and absent from convalescent sera. Drug-dependent bone marrow colony-suppressing activity was removed by absorption on an antiimmunoglobulin-Sepharose column but not by IgG-Sepharose. The serum show non-drug dependent suppression of oxygen consumption by normal polymorphonuclear leukocytes engaged in phagocytosis and also showed evidence of ability to opsonize these cells. When the serum was incubated with mitogen-stimulated lymphocytes, suppression of 3H-thymidine uptake by autologous but not allogeneic cells was noted. Similarly, the serum suppressed short-term 3H-thymidine uptake by autologous but not allogeneic bone marrow. Absorption of the patients' sera with allogeneic polymorphonuclear leukocytes, autologous polymorphonuclear leukocytes, or autologous lymphocytes removed the drug-dependent inhibitory activity, but absorption with allogeneic lymphocytes did not. These data are most consistent with the presence of a noncomplement dependent antibody capable of suppressing granulopoiesis, mediating peripheral destruction of polymorphonuclear leukocytes, and cross-reacting with a lymphocyte antigen of limited population distribution.     

Studies Of The Effects Of Pregnancy Serum On Polymorphonuclear Leukocyte Functions

The phagocytosis of both viable and heat-killed Staphylococcus aureus by normal human neutrophils was diminished in the presence of pregnancy serum. Particle ingestion was reduced significantly (P < 0.0025) after 5 minutes of incubation when leukocytes were in 15% serum obtained during the latter phases of gestation. Control sera from normal adult females or males or from cord blood all functioned normally in support of phagocytosis. Intracellular metabolic events occurring after ingestion, including bacterial killing and nitro-blue tetrazolium reduction, were decreased in proportion to the number of particles ingested. Differential studies involving leukocytes obtained from either pregnant or control subjects, when suspended in either pregnancy or control serum pools, showed the inhibition was due to a humoral and not a cellular factor. Mixing experiments demonstrated that serum obtained during gestation contained a suppressor or neutrophil function rather than a deficiency of a factor required for phagocytosis. The pregnancy serum suppressant affected cells, not the particles ingested, and was found to be non-dialyzable and heat-stable. Hydrocortisone added to control sera to exceed the steroid concentrations found in pregnancy sera did not retard phagocytosis nor diminish bactericidal activity. The data suggest that the active serum component exerts its inhibitory action at the cell surface. Since polymorphonuclear leukocytes are important mediators of inflammation and are implicated in the pathogenesis of tissue destruction, the subsidence of inflammatory diseases during gestation could be related, at least in part, to the inhibitory effects of pregnancy serum on leukocyte functions.     

The Phagocytosis of Blood Leukocytes from Cystic Fibrosis Patients is not Impaired in General

Impaired phagocytosis of Pseudomonas aeruginosa was found in isolated monocytes of peripheral blood of cystic fibrosis patients, but not in their neutrophils, as reported some years ago. In the present study, we analysed the phagocytic capacity of peripheral blood neutrophils and monocytes of cystic fibrosis patients and of healthy controls. Phagocytosis was determined using a commercial phagocytosis “in whole blood” assay on the basis of fluorescence-labelled opsonized Escherichia coli bacteria and flow cytometry. Venous blood of cystic fibrosis patients and of healthy controls was collected and the phagocytosis assay was performed. No differences in the percentage of phagocytic cells or in the overall phagocytic capacity were found between samples of cystic fibrosis patients and healthy controls either in monocytes or in neutrophils. Thus, our results did not support the hypothesis of a generally reduced phagocytic ability in the peripheral blood immune cells of cystic fibrosis patients.

     

Quantitation and identification of antibodies to outer-membrane proteins of Pseudomonas aeruginosa in sera of patients with cystic fibrosis

Chronic, overwhelming pulmonary infection with Pseudomonas aeruginosa is a frequent problem in patients with cystic fibrosis. Titers of antibody to the outer-membrane proteins of P aeruginosa were 10(1)-10(8) (as measured by an enzyme-linked immunosorbent assay) in the sera of 32 patients with cystic fibrosis. Fifteen patients who had been colonized with P aeruginosa for 18 months to nine years had a geometric mean antibody titer of 1.3 X 10(5)--a value approximately 500-fold higher than that for 13 patients with cystic fibrosis who had never been colonized or for 16 healthy adults without cystic fibrosis (P less than 0.001). A significant correlation was observed between the presence of antibody to outer-membrane proteins and the presence of antibody to mucoid exopolysaccharide (P less than 0.002). Nineteen serum specimens from the patients with cystic fibrosis were allowed to react with Western electophoretic blots of separated outer-membrane proteins. All of these sera contained antibodies to porin protein F. In addition, antibodies to outer-membrane proteins E, H2, and I and to a variety of minor protein components were observed in many sera.     

Antibodies to proteases and exotoxin A of Pseudomonas aeruginosa in patients with cystic fibrosis: Demonstration by radioimmunoassay.

Sera from 33 patients with cystic fibrosis and two pediatric patients being treated for chronic pulmonary infections not related to cystic fibrosis and six sera or serum pools from uninfected individuals were tested with a microtiter radioimmunoassay for reactivity against exotoxin A and two proteases from Pseudomonas aeruginosa. Exotoxin A was purified from a low-protease strain of P. aeruginosa and shown to have adenosine diphosphate-ribose transferase activity and mouse lethality. Proteases were purified from an isolate of P. aeruginosa from a patient with cystic fibrosis and had proteolytic activity against elastin and collagen in an assay employing dimethylated protein substrates. The antibody responses of the patients detected using 125I-labeled antibody to human immunoglobulin were correlated with clinical evaluations expressed as a composite score based on pulmonary findings, case histories, growth and nutrition, and chest X rays. Values in the radioimmunoassay for patients' sera were compared with those of a control serum pool and expressed as the ratio of counts per minute (cpm) in patient serum to the cpm in the control pool. Inverse correlations were found between these ratios for each of the pseudomonas exoproducts and clinical scores; highest ratios occurred in patients showing the lowest clinical scores. These results confirm that proteases and exotoxin A of P. aeruginosa are produced in cystic fibrosis pulmonary infections due to P. aeruginosa and suggest that they may serve as significant virulence factors in these chronic infectious states.     

Alveolar macrophage dysfunction in human bone marrow transplant recipients

We studied the functional characteristics of alveolar macrophages obtained by segmental pulmonary lavage from allogeneic marrow transplant recipients without evidence of ongoing pulmonary infection. The macrophages were mostly of donor marrow origin as judged by Y body fluorescence and were morphologically normal, except for the intracellular accumulation of various amounts of heterogeneous foreign materials. Macrophage function of patients studied within four months after transplantation was impaired, as measured by chemotaxis, phagocytosis and killing of Candida pseudotropicalis, and killing of bacteria. In two patients studied six and 12 months after transplantation, macrophage functions returned toward normal except for a persistent defect in killing of C. pseudotropicalis. Cytomegalovirus was cultured from the lavaged cells of two patients, but the macrophage dysfunction was independent of the cytomegalovirus isolation. These results show that alveolar macrophage dysfunction occurs in marrow transplant recipients and may be associated with their increased risk for pulmonary infections.     

The pulmonary disposition of theophylline and its influence on human alveolar macrophage bactericidal function

We studied the pulmonary disposition of theophylline by performing bronchoalveolar lavage on 19 normal, nonsmoking volunteers who had taken theophylline orally for 14 days. In addition, we determined the influence of theophylline on human alveolar macrophage bacterial phagocytosis, intracellular killing, and hydrogen peroxide release. We found a 1:1 relationship between serum and bronchoalveolar lavage theophylline concentrations when lavage fluid concentrations were corrected for saline dilution. We found marked impairment of the bactericidal activity of alveolar macrophages from theophylline-treated subjects (intracellular killing efficiency of 24.7 +/- 1.5% compared with 60.2 +/- 0.9% by macrophages from control subjects; p less than 0.001). This defect in alveolar macrophage bactericidal activity was inversely correlated with the bronchoalveolar lavage theophylline concentrations, and was corrected after the alveolar macrophages were cultured under serum-free conditions for 48 h. Theophylline significantly impaired alveolar macrophage release of hydrogen peroxide. Hence, theophylline may compromise lung host defenses by suppressing alveolar macrophage bactericidal activity and oxidative metabolite release.     

Immunoglobulin-G subclasses in cystic fibrosis. IgG2 response to Pseudomonas aeruginosa lipopolysaccharide

Pulmonary macrophage phagocytosis of Pseudomonas aeruginosa is defective when this pathogen is opsonized with IgG antibodies isolated from serum samples from patients with cystic fibrosis (CF). To evaluate this defect further, IgG subclasses in the serum and lung fluids of patients with CF were quantitated. The pattern of IgG subclasses in serum specimens from patients with CF (n = 15) and in patients without CF but with chronic obstructive airway disease and recurrent P. aeruginosa infection (n = 4) was significantly altered from that found in normal subjects (n = 31). Immunoglobulin-G2 and IgG3 expressed as percentages of total IgG subclasses or in micrograms per milliliter of serum were significantly elevated in the serum specimens of these patients (p less than 0.05), and IgG1 was significantly decreased (p less than 0.01). It appears that the increase in IgG2 in the serum of patients with CF and those without CF but with chronic P. aeruginosa infection may be in response to chronic antigenic stimulation by P. aeruginosa lipopolysaccharide. Evidence presented to support this includes: (1) IgG2 is not increased in CF serum if a history of P. aeruginosa infection is absent, (2) IgG2 levels expressed as percentages of total IgG subclasses in CF lung fluids were positively correlated (r = 0.73) with the number of colony-forming units of P. aeruginosa present in CF sputum specimens, and (3) IgG antibodies specifically eluted from P. aeruginosa lipopolysaccharide ligands on affinity gels were largely restricted to IgG2. The opsonic index, ([IgG3] + [IgG1]) divided by ([IgG2] + [IgG4]), is inverted in CF lung fluids (0.73:1; normal, 2:1). Because pulmonary macrophages show surface receptors binding primarily with IgG3 and IgG1, it may be that such an alteration in IgG subclasses in the respiratory secretions of patients with CF further inhibits opsonin-mediated clearance of P. aeruginosa.     

Neutrophilic alveolitis in idiopathic pulmonary fibrosis. The role of interleukin-8.

Idiopathic pulmonary fibrosis is an immunologically mediated pulmonary disorder in which activated alveolar macrophages (AM) and neutrophils play cardinal roles in the pathogenesis of the inflammatory lung lesion. The factors responsible for the induction and perpetuation of the neutrophilic alveolitis are not known. Recently, a novel cytokine (Interleukin-8) was described that is released by activated mononuclear phagocytes and a variety of other cell types, and it exhibits potent chemotactic activity for polymorphonuclear leukocytes (PMN). Increased expression of IL-8 has been described in other inflammatory disorders characterized by neutrophilic infiltration, including psoriasis, rheumatoid arthritis, and the sepsis syndrome, but no studies have assessed this cytokine in the context of interstitial pulmonary disorders. We have previously shown that normal human AM release IL-8 upon appropriate stimulation, but data assessing the expression of IL-8 by human AM in specific pulmonary disease states are lacking. In this study, we examined the expression of steady-state mRNA for IL-8 by human alveolar macrophages obtained by bronchoalveolar lavage (BAL) from patients with idiopathic pulmonary fibrosis (IPF) or sarcoidosis and from healthy volunteers. Because it is known that adherence to plastic culture plates may up-regulate gene expression for IL-8 in the absence of additional stimulation, we extracted mRNA immediately from the cell pellet obtained by BAL rather than using cultured alveolar macrophage monolayers. Northern blot analysis was performed to determine IL-8 mRNA expression. We found that BAL cells from patients with IPF constitutively expressed mRNA for IL-8, and the amount of IL-8 mRNA (as assessed by laser densitometry) correlated with the percent of neutrophils on BAL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low sweat electrolytes in a patient with cystic fibrosis

A patient with the clinical syndrome of cystic fibrosis characterized by chronic pulmonary disease, infection with mucoid Pseudomonas aeruginosa, sinusitis, nasal polyposis, abnormal pancreatic bicarbonate response to secretin stimulation, but normal levels of trypsin and chymotrypsin in the duodenal drainage, and a sibling with autopsy-documented cystic fibrosis, is described. Sweat chloride ranged from 20 to 44 meq/liter and sweat sodium from 36 to 55 meq/liter. Immunoglobulin deficiency, α₁-antitrypsin deficiency, tuberculosis and abnormalities of ciliary ultrastructure were excluded. Review of sweat electrolytes in 213 patients with cystic fibrosis revealed that patients with normal pancreatic enzyme release have significantly lower sweat sodium and chloride concentrations (p <0.0005) than do patients with pancreatic insufficiency.Chronic pulmonary disease, pancreatic insufficiency and elevated levels of sweat electrolytes comprise the classic diagnostic triad for cystic fibrosis. The expression of these features may be variable, but the sweat test remains the cardinal laboratory confirmation of the diagnosis. Over 98 percent of patients with cystic fibrosis have sweat chloride values greater than 60 meq/liter, 1 to 2 percent between 50 and 60 meq/liter, and only about one in 1,000, like our patient, less than 50 meq/liter. Patients with cystic fibrosis with borderline sweat chloride values frequently have chronic pulmonary disease but intact pancreatic enzyme release. In such patients, family history, ancillary clinical features and systematic exclusion of other syndromes assume special diagnostic importance.     

Elevation of Anti-cytokeratin 19 Antibody in Sera of the Patients With Idiopathic Pulmonary Fibrosis and Pulmonary Fibrosis Associated With Collagen Vascular Disorders

It has been suggested that cytokeratin 19 is expressed in regenerated bronchoepithelial cells in patients with pulmonary fibrosis, and serum cytokeratin 19 fragment is elevated in patients with pulmonary fibrosis. We hypothesized that serum antibodies to cytokeratin 19 may be formed in patients with pulmonary fibrosis. To prove the existence of anti-cytokeratin 19 antibodies in patients' sera, human recombinant cytokeratin 19 was stained with patients' sera by a Western immunoblot. Then, we tried to establish an enzyme-linked immunosorbent assay to quantitate anti-cytokeratin 19 antibody in the sera of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis associated with collagen vascular disorders (PF-CVD). We demonstrated the anti-cytokeratin 19 antibody in patient' sera by a Western immunoblot. In patients with IPF and PF-CVD, significantly high anti-cytokeratin 19 antibody was demonstrated compared with normal volunteers, patients with chronic bronchitis, and patients with pneumonia. These results suggest that anti-cytokeratin 19 antibody may have played a role in the process of lung injury in pulmonary fibrosis. Accepted for publication: 27 May 1999     

The immunology of cryptococcal disease

Cryptococcus neoformans is a ubiquitous fungus found in the soil. Upon inhalation, a complex, incompletely understood series of host responses begins that determines whether the infection will be controlled or will progress to local or disseminated disease. Local pulmonary disease may be asymptomatic or may pursue a subacute course with mild pulmonary symptoms and systemic complaints suggestive of tumor. In the compromised host, however, symptomatic pulmonary disease is often the harbinger of systemic dissemination. Early host responses include phagocytosis by polymorphonuclear leukocytes aided by complement activation which provides opsonins. Lymphocytes are activated to produce lymphokines which may enhance macrophage phagocytosis and intracellular killing of ingested cryptococci. Other lymphocytes may function as natural killer-like cells or inhibit the growth of the fungus. Production of antibody further facilitates phagocytosis by both polymorphonuclear leukocytes (PMN) and monocytes (MC). In the presence of antibody, both PMN and MC demonstrate antibody-dependent cell-mediated cytotoxicity. The combination of humoral and cell-mediated immunity in normal hosts appears to provide excellent protection against disseminated infection as evidence by the rarity of disease in exposed individuals with positive skin tests. By contrast, the frequency of cryptococcal disease in steroid-treated individuals, allograft recipients, and AIDS victims highlight the importance of T lymphocyte dependent host defenses. In view of compelling in vitro evidence for the importance of humoral responses, the infrequency of cryptococcal disease in patients with gammopathies remains a puzzle.     

Increased levels of interleukin-1 in bronchoalveolar washings from children with bacterial pulmonary infections

To investigate its role in pulmonary infections, concentrations of interleukin-1 were measured in 22 bronchoalveolar lavage fluid (BALF) samples from 19 children with cystic fibrosis (CF), and in 13 disease controls by enzyme-linked immunosorbent assay (ELISA) for IL-1 beta and the D10.G4.1 proliferation assay for IL-1 activity. Significantly higher levels of IL-1 beta and IL-1 activity were found in BALF from patients with bacterial pulmonary infections than in those without such infection. There was no significant difference between the levels in patients with CF and pulmonary infections and those in children with bacterial infections complicating other diseases. High performance liquid chromatography showed that most of the IL-1 beta was associated with a molecular weight peak of 17 to 18 kD. Pulmonary inflammation reflected by the number of polymorphonuclear leukocytes (PMN) in the sample correlated significantly with the IL-1 concentration.     

Oxidative changes of bronchoalveolar proteins in cystic fibrosis

Chronic bacterial infection and severe, polymorphonuclear neutrophil-dominated endobronchial inflammation are characteristic hallmarks of cystic fibrosis (CF) lung disease. The free radicals generated can be deleterious for structure and function of many proteins. The goal of this study was to investigate the degree of oxidation of pulmonary epithelial lining fluid proteins. BAL fluid (BALF) from 55 children with CF and from 11 patients in a control group were investigated by dot-blot assay for content and by two-dimensional electrophoresis and Western blotting for the pattern of distribution of oxidized proteins. The highest level of oxidative stress, as assessed by the level of protein carbonyls, was found in patients with FEV1 < 80% of predicted or with highly elevated neutrophil counts. Compared to control subjects without lung disease, CF patients with normal lung function and CF patients with a normal neutrophil count in their BALF had significantly higher protein carbonyl levels. The extent of protein oxidation was directly related to the neutrophil granulocyte count and inversely to lung function. Our data support the hypothesis that oxidative damage of pulmonary proteins during chronic and excessive neutrophilic endobronchial inflammation may contribute to the decline of lung function in CF patients.     

Participation of immunoglobulin and the alternative complement pathway in opsonization of Bacteroides fragilis and Bacteroides thetaiotaomicron

Studies were conducted to determine the requirements for immunoglobulin and complement in opsonization of Bacteroides fragilis and Bacteroides thetaiotaomicron. The ability of human sera depleted of immunoglobulin or of components of complement to promote the phagocytosis and intracellular killing of the two strains of Bacteroides by human leukocytes was measured in vitro under anaerobic conditions. Neither hypogammaglobulinemic sera nor pooled normal human serum that was heated at 56 C for 30 min supported phagocytosis and killing of the two strains of Bacteroides. Neither sera depleted of terminal complement components by treatment with inulin or cobra venom factor nor human serum deficient in C8 supported phagocytosis of the tested strains. In addition, pooled normal human serum depleted of C3, factor B, or factor D did not support phagocytosis of either strain. Dose-dependent restoration of the opsonic activity of factor B-depleted serum was accomplished by purified human factor B but not by human C2. The results indicate that immunoglobulin and components of the alternative comed in this study.     

Selective immunoglobulin M (IgM) deficiency in two immunodeficient adults with recurrent staphylococcal pyoderma.

Two adult men with recurrent pyoderma due to Staphylococcus aureus and a selective deficiency of immunoglobulin M (IgM) antibody synthesis are described. An analysis of each patient's polymorphonuclear leukocyte chemotaxis, phagocytosis and killing of Staph. aureus, serum opsonizaiton of Staph. aureus, and serum and lymphocyte-mediated responses to antigenic stimulation was performed. Family studies revealed a possible autosomal dominant inheritance pattern with heterogenetic expression of various dysgammaglobulinemic states in each patient's first degree relatives. In vivo studies of delayed hypersensitivity and in vitro studies of polymorphonuclear leukocyte and lymphocyte function were normal. A defect in IgM, but not in IgG (immunoglobulin G), antibody synthesis to a number of antigens, and a mild decrease in serum opsonic activity to Staph. aureus correctable by heat inactivated normal human serum were found in each patient. In these patients, the recurrent staphulococcal pyoderma prompted an investigation of host defense mechanisms and revealed low to absent IgM levels and a defect in IgM antibody synthesis.     

Eradication of methicillin-resistant Staphylococcus aureus from the lower respiratory tract of patients with cystic fibrosis

Two of 95 patients followed in an adult cystic fibrosis clinic consistently grew methicillin-resistant Staphylococcus aureus (mrsa) on sputum culture. Sputum Gram stain consistently showed +4 polymorphonuclear leukocytes and +4 Gram-positive cocci in clusters. Both patients were co-infected with Pseudomonas aeruginosa and required multiple hospitalizations for treatment of pulmonary exacerbation, resulting in significant infection control concerns. Multiple courses of antibiotics, including ciprofloxacin and clindamycin regimens, failed to eliminate the mrsa. A combination of oral rifampin and clindamycin was successful in eradicating the organism from both patients. Over a 12-month period following therapy, in both patients none of 13 sputums showed Gram-positive cocci in clusters on Gram stain and none of 13 sputum cultures grew mrsa. Successful eradication of mrsa has greatly simplified infection control measures on subsequent hospitalizations, reducing costs and enhancing patient comfort.     

Genetic deficiency of C4 presenting with recurrent infections and a SLE-like disease: Genetic and immunologic studies

A young girl presenting with recurrent pulmonary infections and atypical lupus erythematosus was totally deficient in C4. In one sister, also deficient in C4, the same symptoms developed. Results of family studies were consistent with an autosomal recessive mode of transmission and with linkage of the genes determining C4 deficiency to those of the major histocompatibility complex. The patient's serum and red cells were Chido- and Rodgers-negative. Humoral and cellular immunity were normal, except for a low lymphocyte response in mixed lymphocyte culture. The cellular function of the patient's polymorphonuclear leukocytes was normal, for both phagocytosis and bactericidal activity using Candida albicans. However, in the presence of C4-deficient serum, opsonin generation and bactericidal indexes were diminished. These defects were completely reversible upon addition of purified C4.     

Assessment of early alveolar particle clearance and macrophage function following an acute inhalation of sulfuric acid mist

Rabbits were exposed to submicrometer sulfuric acid mist at 1 mg/m3 for 1 hr to assess effects on alveolar region clearance of a polystyrene latex tracer aerosol. Bronchopulmonary lavage was performed at selected times after exposure for functional characterization of alveolar macrophages. In vivo, clearance was accelerated in acid exposed animals relative to sham controls. Acid exposure produced no change in the viability or numbers of macrophages recovered. Although an increase in the number of polymorphonuclear leukocytes, primary neutrophils, was observed by 1 hr in both acid and sham groups, compared to nonexposed controls, levels were normal by 12 hr in shams but continued elevated in the acid group through 24 hr. Reduced in vitro macrophage adherence was observed after acid exposure. In vivo uptake of the tracer particles by macrophages was enhanced during the first 3 hr after acid exposure and in vitro phagocytosis by polymorphonuclear leukocytes was increased through 48 hr post-exposure. The results indicate some functional alterations in free cells after in vivo exposure to H2SO4 and the production of a mild inflammatory response. This latter was associated with an acceleration of inert particle clearance from the alveolar region.     

阿奇霉素对肺间质纤维化大鼠干预作用的研究

目的 探讨阿奇霉素对博莱霉素所致大鼠肺间质纤维化的干预作用及机制。方法 雄性Ｗｉｓｔａｒ大鼠４２只,分为３组。博莱霉素模型组：１８只,大鼠气管内一次性注入博莱霉素５ｍｇ／ｋｇ,制成肺间质纤维化模型,阿奇霉素治疗组：１８只,模型制备同博莱霉素模型组,模型制备２小时后,顷胃管注入阿奇霉素８０ｍｇ／ｋｇ,每日１次,每周连续３日。两组动物于１、２和４周处死。对照组：６只,大鼠气管内注入生理盐水０．５ｍｌ,