Richard D. deShazo

Mechanisms of neutrophil damage to human alveolar extracellular matrix: The role of serine and metalloproteases☆

Many syndromes of lung injury are associated with accumulation of neutrophils within the pulmonary parenchyma. These neutrophils have the capacity to produce lung injury by products including proteases and reactive oxygen species (ROS). We examined the ability of activated neutrophils to solubilize human alveolar extracellular matrix (ECM), and by use of scavengers and inhibitors, evaluated the role of ROS and proteases in this process. Supernatants of phorbol myristate acetate-activated neutrophils routinely solubilized 10.2% +/- 0.8% (n = 30) of collagen in human alveolar ECM, as measured by hydroxyproline release. Scavengers of ROS had no significant effect on ECM solubilization. Inhibitors of metalloproteases partially inhibited ECM solubilization (38.5% +/- 4.6% inhibition by ethylenediaminetetraacetic acid [n = 6], and 37.0% +/- 14.7% by 1,10-phenanthroline [n = 6]; p less than 0.05). Inhibitors of the neutrophil serine proteases, elastase and cathepsin G, markedly inhibited ECM solubilization (100.9% +/- 3.7% by alpha 1-protease inhibitor [alpha 1-PI] [n = 6] and 81.9% +/- 0.1% by soybean trypsin inhibitor [n = 6]; p less than 0.01). Since alpha 1-PI completely inhibited solubilization, metalloprotease activity appeared to be related to serine protease activity. This finding was confirmed by the observation that addition of a metalloenzyme activator, p-aminophenylmercuric acetate, in the presence of alpha 1-PI, restored solubilization to the same level as that inhibited by metal chelators. We conclude that human neutrophil metalloproteases and serine proteases directly solubilize human alveolar ECM. Furthermore, neutrophil serine proteases activate latent metalloproteases. However, ROS were not demonstrated to play a major role in ECM solubilization in our system.

Current concepts about the pathogenesis of silicosis and asbestosis

Silicosis and asbestosis are two forms of fibrotic lung disease resulting from the inhalation of inert materials indigestible by pulmonary alveolar macrophages. Results of studies of the host response to these particulates have not always been consistent. It is clear, however, that after phagocytosis, both cause alveolar macrophage damage, with resultant release of macrophage products, including fibrogenic factors and chemotactic factors for neutrophils. The latter cells also release lysosomal enzymes and free radicals when exposed to silica and asbestos. The net effect of these observations suggests that the combination of tissue damage and fibroblast stimulation results in the pulmonary fibrosis characterizing these diseases. Patients with silicosis and asbestosis have normal or decreased cell-mediated and increased humoral immunity with a high incidence of circulating immune complexes and autoantibodies. Whether these abnormalities are related to the pathogenesis of pulmonary fibrosis or are epiphenomena remains to be determined.

A modified double antibody sandwich enzyme-linked immunosorbent assay for measurement of alpha-1-antitrypsin in biologic fluids☆

Alpha-1-antitrypsin (alpha 1AT) is the major protease inhibitor in human serum, and plays an important role protecting tissues from potentially harmful enzymes released during inflammatory reactions. Proteolytic enzymes such as leukocyte elastase are usually released and inactivated locally at the site of inflammation, so there has been much recent interest in measuring local alpha 1AT concentrations in biologic fluids. In this study, we developed a modified double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and used it to measure alpha 1AT concentrations in several biologic fluids. The assay was sensitive to as little as 20 ng/ml of alpha 1AT. Serum concentrations measured by the ELISA correlated well with levels determined by radial immunodiffusion (RID) and the ELISA was far more sensitive than RID. In synovial fluid, higher concentrations determined by the ELISA compared with RID probably reflect interference of diffusion of alpha 1AT in the RID gel by hyaluronic acid and protease-inhibitor complexes. Synovial fluid did not interfere with the detection of added alpha 1AT by ELISA, but it did reduce the amount detected by RID by about 30% in 2 fluids. In saliva, alpha 1AT concentrations of less than 1 microgram/ml were easily quantified. Bronchoalveolar lavage fluids have been extensively studied because of the important role of alpha 1AT in pulmonary inflammatory processes. We found concentrations of 1-3 micrograms/ml in most samples with our assay. These levels were comparable to those previously reported with assays that required up to 50-fold concentration of the fluid. Neither saliva nor bronchoalveolar fluid significantly interfered with detection by ELISA of added alpha 1AT. This modified double antibody sandwich ELISA may have broad applications for studies of the role of alpha 1AT in health and disease.

Immune abnormalities associated with HLA-B8: lymphocyte subsets and functional correlates

Mononuclear cell populations were enumerated in healthy young adults with or without the histocompatibility antigen HLA-B8. Mononuclear cell counts were lower in subjects with HLA-B8, as was reflected in lower absolute numbers of the cell subsets. When cell populations were compared as percentages of total mononuclear cells, subjects with HLA-B8 had significantly more B lymphocytes bearing IgM than did subjects without HLA-B8. The T4/T8 ratio was significantly increased in subjects with HLA-B8. due both to increases in OKT4+ cells and to decrease in OKT8+ cells. B-Lymphocyte function was compared in subjects with and without HLA-B8 by measuring pokeweed mitogen driven differentiation of B cells to IgM bearing blasts, and was significantly greater in subjects with HLA-B8. Subjects with HLA-B8. Subjects with HLA-B8 were also found to have decreased Con A-induced suppression. Alterations in lymphocyte subsets and lymphocyte functions may underlie the predisposition to autoimmunity associated with HLA-B8.

Immunologic assessment of a cluster of asymptomatic HTLV-I-infected individuals in New Orleans

PURPOSEnAlthough clusters of individuals infected with the human T-cell lymphotrophic virus type I (HTLV-I) have been identified in the United States, no systematic evaluation of the immunologic status of these persons has been reported. We therefore studied a group of 11 HTLV-I-infected former intravenous drug abusers who were long-term participants in a methadone maintenance program in New Orleans, Louisiana, to determine the effects of HTLV-I and chronic opiate use on immunity.nnnPATIENTS AND METHODSnMitogenic responses and results of serologic studies, cell phenotype analysis, and cytotoxicity assays were compared to those in two other HTLV-I seronegative groups: a similar group of 17 methadone users and 15 healthy age-, sex-, and race-matched control subjects. All study participants were seronegative for human immunodeficiency virus type 1.nnnRESULTSnPercentages and numbers of total T lymphocytes (CD2+,CD3+), T-suppressor/cytotoxic lymphocytes (CD8+), cytotoxic lymphocytes (Leu7+, Leu11+, NKH-1+) and B lymphocytes (B4+) were similar among the study groups. Although percentages and numbers of total T-helper lymphocytes (CD4+) were also similar among the groups, HTLV-I-infected subjects had higher percentages and proportions of helper/inducer cells (CD4:4B4+) than did HTLV-I seronegative methadone users. Both methadone using groups had decreased percentages and numbers of suppressor/inducer T lymphocytes (CD4:2H4+). Major histocompatibility complex unrestricted T-cell cytotoxicity (lectin-dependent cellular cytotoxicity), natural killer cell function, and mitogenic responses to the T-cell mitogen phytohemagglutin were similar among the three study groups. Pokeweed mitogen responses were severely depressed in the HTLV-I-infected population.nnnCONCLUSIONSnWe conclude that HTLV-I infection is associated with abnormalities in T-cell-dependent B-cell proliferative responses. Furthermore, both long-term methadone use and HTLV-I infection are associated with abnormalities in the distribution of CD4+ cell subpopulations. The increase in the helper/inducer and T-cell cell populations and decrease in the pokeweed mitogenic response noted in HTLV-I-infected subjects appear to be markers for infection with this retrovirus.

Immunologic aberrations in asbestos cement workers: dissociation from asbestosis

Immunoregulatory disorders have been implicated in the pathogenesis of asbestosis. We therefore compared the immunologic status of a well-characterized group of 31 current and former asbestos-cement workers with that of a group of 52 healthy controls, after adjustments had been made for the possible confounding effects of age, race, and smoking. The asbestos workers had significantly decreased percentages and numbers of both B and T lymphocytes in peripheral blood and a paradoxical IgG hypergammaglobulinemia. Analysis of T-lymphocyte subpopulations revealed that total T-cell numbers (OKT3+), helper-inducer T-cell numbers (OKT4+), and suppressor-cytotoxic T cell numbers (OKT8+) were decreased by similar proportions. These decreases were negatively correlated with time elapsing since the end of exposure to asbestos. In both workers and controls, lymphocyte proliferative responses to phytohemagglutinin (PHA) were correlated positively with the number of OKT4+ cells and negatively with age and serum IgG levels. When adjustments had been made for these confounding variables, no differences in PHA responses were noted between workers and controls. No relationship was detected in the workers between any of the immunologic aberrations noted and (1) radiographic category of pneumoconiosis, (2) estimates of cumulative asbestos exposure, or (3) abnormalities of pulmonary function. These data suggest that the immunologic perturbations we have noted in asbestos-exposed individuals are epiphenomena, unrelated to the pathogenesis of asbestosis itself.

Analysis of depressed cell-mediated immunity in asbestos workers

To explore the mechanisms of asbestos-related perturbations of the immune system, we evaluated the in vitro cell-mediated immunity of five asymptomatic asbestos workers with hypergammaglobulinemia and decreased T-cell numbers. These results were compared with those in 10 matched controls. Analysis of T-lymphocyte populations revealed decreased absolute numbers of OKT4+ (helper/inducer) T cells in the peripheral blood and phytohemagglutinin (PHA)-stimulated mononuclear cell cultures of the workers. When chrysotile asbestos was added to PHA cultures, expansion of OKT4+ cell populations was disproportionately inhibited in workers cultures. Furthermore, control proliferative responses to PHA became indistinguishable from initial worker responses. These effects were incompletely explained by the cytotoxic effects of asbestos on cultured lymphocytes. We conclude that both in vivo and in vitro exposure of mononuclear cell populations to asbestos may lead to a diminution of helper-inducer T-cell numbers. In asbestos-exposed individuals, this latter lymphocyte subpopulation appears to be especially sensitive to in vitro asbestos exposure. Although the clinical implications of these findings are unclear, we hypothesize that many of the immunologic abnormalities that occur in asbestos workers could be explained by direct asbestos effects on the OKT4+ immunoregulatory population.

Murine alveolar macrophage-mediated lymphocyte cytostasis: kinetics and mechanisms.

Abstract Recent reports have demonstrated that alveolar macrophages (AM) from several species regulate antigen- and mitogen-induced blastogenesis. In this study, we confirm that murine AM also mediate lymphocyte cytostasis and define, in part, the mechanism involved. AM were found to inhibit homologous splenocyte responses to concanavalin A in a dose-dependent manner. The inclusion of 1 AM:10 lymphocytes abrogated mitogenesis. Kinetic studies revealed that maximal inhibition of the splenocyte response required the inclusion of AM at culture initiation, stimulation of splenocytes with an optimal Con A dose, and an optimal incubation period of 72 hr. In addition, suppression of Con A-induced blastogenesis by AM was not genetically restricted, as Balb/c AM suppressed allogeneic CBA/J spleen cells comparably to homologous control cells. The addition of either catalase or indomethacin to partially suppressed cultures (containing 3% AM) totally reversed the inhibition. In contrast, catalase did not protect lymphocytes from absolute suppression mediated by higher AM numbers (10% AM), while indomethacin offered partial protection. A synergistic effect was noted upon the addition of both substances. Thus, prostaglandin and hydrogen peroxide released by AM contribute to the suppressive effects of these cells.

Evidence for histamine-mediated inhibition of monocyte chemotaxis in atopic dermatitis.

Leukocyte chemotaxis was studied in 11 patients with severe childhood onset atopic dermatitis at a time when their disease was relatively quiescent. Pyoderma had been an important complication of the dermatitis in these patients. The chemotactic responsiveness of patient neutrophils and monocytes was on the average not significantly different from that of healthy control subjects, although three patients were identified who had significantly impaired responses. No correlation between IgE levels and leukocyte chemotaxis was observed. Because excessive amounts of histamine have been recovered from the skin of patients with atopic dermatitis, we evaluated the effects of histamine on the chemotactic responsiveness of leukocytes from these patients. Histamine caused a small dose-related increase in chemotaxis of neutrophils from both patients and control subjects (10(-7)M to 10(-5)M histamine). In contrast, histamine had no effect on the chemotaxis of monocytes from control subjects but inhibited the chemotactic responsiveness of monocytes from atopic dermatitis patients. These findings suggest that an abnormal sensitivity of monocytes to histamine is an intrinsic feature of atopic dermatitis that may be detectable when the disease is quiescent. Furthermore, this abnormality may contribute to the impairment of monocyte chemotaxis that has been previously observed in patients with active atopic dermatitis.

Reliability of Cell Counts and Protein Determinations in Serial Bronchoalveolar Lavage Procedures Performed on Healthy Volunteers

We measured the variability in volume, total cells, cell types, and proteins in the bronchoalveolar lavage fluid recovered from 10 volunteers (five smokers, five non-smokers) lavaged repeatedly over a three-year period. Thirty lavages were performed using a rigorously standardized approach. Differential counts on the cytospin preparations were performed by three independent readers and interobserver variability in the interpretation of these counts measured. Variability in interpreting the cellular counts was less in smokers than non-smokers and decreased as the number of cells of any particular type increased. Only one reader interpreting the mean percentage of cells recovered of one cell type (neutrophils) in only one smoking group, the nonsmokers, was significantly different from the other two. There was also considerable variability in bronchoalveolar lavage fluid total protein, albumin, IgG, and IgA. Expressing albumin and IgG as a percentage of total protein recovered and expressing IgA and albumin as a ratio in nonsmokers lessened the variability of these parameters. Mean and standard deviations of the cellular and protein concentrations showed that large differences in these parameters would be necessary in order to attribute these changes to changes in the underlying pulmonary status. Excessive variability in nearly all parameters in this group without recognized lung disease challenges the usefulness of this test in the clinical assessment of patients serially followed because of underlying lung disease.