Brian E. Bozelka

An Immunologic Evaluation of Hemophiliac Patients and Their Wives: Relationships to the Acquired Immunodeficiency Syndrome

Recently, hemophiliac patients receiving factor VIII concentrate therapy have developed the acquired immunodeficiency syndrome. Because abnormalities of cell-mediated immunity are found in this syndrome, we evaluated the peripheral blood immunologic status of 24 patients with classic hemophilia and 5 patients with factor IX deficiency. Both groups had decreased percentages and numbers of helper-inducer lymphocytes (OKT4+) and increased percentages of suppressor-cytotoxic T-lymphocytes (OKT8+) which resulted in depressed OKT4/T8 ratios. Abnormalities of the T-lymphocyte subpopulation were most severe in 7 patients with factor VIII deficiency with lymphadenopathy who also had increased Ia+ cells and profoundly suppressed lymphocyte mitogenic responses. No correlation was found between OKT4/OKT8 ratios or lymphocyte responses to mitogen and the amount of factor VIII concentrate used per year. Evaluation of five wives of factor-VIII-deficient patients who had abnormal OKT4/OKT8 ratios showed decreased percentages of OKT4 cells, but normal lymphocyte mitogenic responses. Serum levels of IgG were elevated in factor-VIII-deficient patients but not their wives or factor-IX-deficient patients. We conclude that T-lymphocyte subpopulation abnormalities and lymphocyte mitogenic responses are depressed in asymptomatic hemophiliac patients receiving either factor VIII or factor IX concentrates. These abnormalities are most severe in otherwise asymptomatic hemophiliac patients who have developed lymphadenopathy.

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.

Humoral immunologic abnormalities in workers exposed to asbestos cement dust

Serum specimens from 144 workers exposed to asbestos cement dust were examined for the presence of ANA, RF, immunoglobulins, and IC. These immunologic findings were compared with chest radiographic changes. A high percentage of workers had polyclonal hypergammaglobulinemia, and there was a statistically significant association between elevated levels of IgG and IgM and radiographic classification. Although a significant number of workers had an increased prevalence of ANA and elevated levels of IC, there was no correlation between these parameters and chest radiographs. These findings support B cell hyperactivity in workers exposed to asbestos and suggest that autoantibody production and IC are not directly involved in disease pathogenesis.

The Immunoregulatory Nature of Iron. II. Lymphocyte Surface Marker Expression

Previously, we presented preliminary evidence that supported our hypothesis for the immunoregulatory nature of iron [7]. The objective of the present work was to test that hypothesis in greater detail. Our approach was to examine the effect that iron had on the expression of the surface markers on lymphocytes that had been activated by pokeweed mitogen (PWM). The two categories of lymphoid surface molecules were enumerated on those cells; first were those that identify T lymphocytes and second, those that appear on the membrane of T cells following activation. The results, as regards T cell‐associated molecules, demonstrated that iron suppresses the expression of the molecules identified by the monoclonal antibodies OKT3 and OKT4. It suppressed expression of the T4 molecule in PWM‐activated cells (30.6% ± 4.5; n = 5) compared with untreated but activated cells (52.2% ± 2.9; n = 5; P = 1.9 × 10−3) resulting in a reduced helper:suppressor T cell ratio from 2.2 ± 0.4 to 1.2 ± 0.3. With regard to activation‐associated lymphocyte markers, iron significantly enhanced expression of the receptor for transferrin as identified by the monoclonal antibody, OKT9. However, it failed to change significantly the expression of three other activation‐associated markers, namely, la, T10, and the receptor that forms thermostable erythrocyte‐rosettes (TE‐R) with sheep red blood cells (SRC). We conclude from those results that iron has a differential immunoregulatory influence on the expression of certain lymphocyte surface molecules on actively dividing lymphocytes.

Asbestos-induced alterations of human lymphoid cell mitogenic responses

Using mitogenic assays, we have investigated the short term effects of two asbestos (amosite and chrysotile) fibers on lymphocyte functions in vitro. These oppositely charged fibers produced different alterations in mitogenesis. The blastogenic responses of concanavalin-A (Con-A) and pokeweed mitogen stimulated human peripheral blood mononuclear cells (PBMN) were significantly increased by the inclusion of 6 micrograms of chrysotile to the culture media. Amosite fibers proved to be inhibitory in all tests. When PBMN were depleted of monocytes, asbestos-related alterations of Con-A responsiveness were unchanged among the remaining cells. However, the addition of chrysotile to phytohemagglutinin (PHA) cultures resulted in a significant increase of the mitogenic response. When PBMN were enriched for T lymphocytes, and again cultured with the mitogens and fibers, the Con-A response still displayed impressive enhancement with chrysotile. In contrast to an intact PBMN population, PHA-induced blastogenesis among these T-enriched lymphocytes was significantly elevated. These experiments demonstrate that asbestos can induce significant changes in the functional integrity of PBMN following a relatively short exposure time in culture.

The Immune System in Hereditary Hemochromatosis: A Quantitative and Functional Assessment of the Cellular Arm

The objective of this investigation was to evaluate certain quantitative and functional characteristics of the effector cells of the cellular arm of the immune system in hereditary hemochromatosis (HH) with respect to treatment status. Two observations were consistent with the postulate that the elevated levels of storage iron has in vivo immunoregulatory properties: (1) the absolute number of CD8-positive T cells were significantly elevated in untreated HH patients (n = 7) and reduced in treated patients (n = 7), as compared with controls; and (2) the proliferative response of peripheral blood mononuclear cells from untreated HH patients to mitogens was suboptimal but the response of peripheral blood mononuclear cells (PBM) from treated HH patients was normal. Furthermore, immunoglobulin secretion by PBM from treated HH patients as compared to controls was altered. Finally, one T effector cell abnormality was unrelated to treatment status in that a subset of mature, non-activated T lymphocytes aberrantly formed thermostable erythrocyte-rosettes (TE-R), a lymphoid surface marker usually expressed on thymocytes or activated T cells. Taken together these data define certain immune alterations that are consistent with the interpretation that cellular immunity may be influenced by the high level of storage iron in HH patients.

Inhibition of mixed leukocyte culture responses in cadmium-treated mice

Previous studies have documented inhibition of antibody-mediated immunity by exposure of experimental animals to heavy metals. To date, very little is known of possible effects of heavy metal ions on cellular-mediated immunity. The mixed leukocyte culture (MLC) assay was used in this study to evaluate the effects on lymphocyte blastogenic response of chronic exposure of mice to cadmium (2 mg CdCl2/kg body wt/day). Spleen cells from two strains of mice treated with CdCl2 were unable to respond as effectively as control normal spleen cells to stimulation with allogeneic leukocytes. Furthermore, X-irradiated splenocytes from the cadmium-stressed animals were less effective than splenocytes from control normal mice as stimulator cells in the MLC reaction. This comparative decrease in T-lymphocyte reactivity could not be attributed to loss of lymphocyte viability or to decrease in ability of lymphocytes from cadmium-treated animals to incorporate tritiated thymidine. Although spleen cells from cadmium-intoxicated mice responded poorly to stimulation with allogeneic leukocytes, they were capable of responding to a mitogenic stimulus. Possible explanations for these observations are discussed.

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.

Thermostable erythrocyte rosette-forming lymphocytes in hereditary hemochromatosis. I. Identification in peripheral blood

Although the immunoregulatory role of iron has been demonstratedin vitro, evidence for a similar rolein vivo is controversial. We have, therefore, studied certain functional and structural properties of lymphocytes in hereditary (idiopathic) hemochromatosis (HH), a disease characterized by iron overload. T- and B-lymphocyte percentages in peripheral blood, serum immunoglobulin levels, and proliferative responses of peripheral blood mononuclear cells (PBM) to lectins were comparable with those of controls. Furthermore, HH serum with elevated iron concentrations did not significantly alter proliferative responses of normal lymphocytes to mitogens. In contrast to those normal findings was the identification of a subset of T lymphocytes in HH that formed rosettes with sheep red blood cells (SRC) at 37°C in abnormally high numbers. Those lymphocytes that formed thermostable erythrocyte rosettes (TE-R) were not immature thymocytes, activated T lymphocytes, or an artifact of passive attachment of anti-SRC antibodies to the HH lymphocyte surface. Their presence did not correlate with a concentration of iron in the serum, the length of treatment, or the presence of the HLA antigen, A3. We conclude that the cellular expression of HH may be detected not as an immunological abnormality, but rather as an abnormality in receptor expression.

Asbestos-Induced Alteration of Human Peripheral Blood Monocyte Activity

Incubation of chrysotile and anthophyllite asbestos fibers with normal human peripheral blood monocytes resulted in significant suppression of monocyte metabolic activity as measured by chemiluminescence. Both fiber types were cytotoxic to monocytes and depressed monocyte phagocytosis of latex beads. We conclude that asbestos-induced monocyte cytotoxicity could result in release of lysosomal enzymes and/or degradation products which contribute to fibrosis in asbestosis. The depression of phagocytosis and microbicidal function may contribute to the increased incidence of carcinogenesis observed in asbestosis.