Decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients.

Anti-inflammatory substances are released during septic shock that modulate monocyte function. Decreased monocyte responsiveness to bacterial toxins and decreased expression of human-leukocyte-associated antigen-DR (HLA-DR) have been reported during septic shock and critical illness. Impaired antigen presentation has been inferred from these observations but has not been demonstrated. We assessed antigen presentation and costimulatory molecule expression in 12 age-matched control subjects, 10 noninfected critically ill patients (CINS), and 17 critically ill patients with sepsis (CIS). Antigen presentation was assessed by using in vitro lymphocyte 5-bromo-2-deoxyuridine (BrdU) incorporation in response to tetanus toxoid. The expression of HLA-DR and the costimulatory molecules CD28, CD86, and CTLA-4 was assessed by flow cytometry. Serum interleukin-10 (IL-10) was also measured by enzyme-linked immunosorbent assay. Serum IL-10 levels were significantly elevated in CIS patients (91 +/- 38 pg/mL) as compared with levels in control subjects (5 +/- 4 pg/mL)(P < .05). Lymphocyte BrdU incorporation increased by 710% +/- 243% in control subjects but by only 144% +/- 62% in CIS patients and 76% +/- 31% in CINS patients (P < .01 vs control). Monocyte HLA-DR expression, monocyte CD86 expression, and lymphocyte CD28 expression were significantly decreased in CIS patients (P < .01) as compared with control subjects. Conversely, lymphocyte CTLA-4 expression was significantly increased in CIS patients (P < .05 vs control). Monocyte CD86 expression was also significantly decreased in CINS patients as compared with control subjects. These data indicate that antigen presentation is decreased in critically ill patients with sepsis. This appears in part related to decreased expression of HLA-DR and the costimulatory molecules CD86 and CD28. Increased expression of the negative signal receptor CTLA-4 may also impair antigen presentation in patients with sepsis.

Immunization with bacillus Calmette-Guerin vaccine increases aortic atherosclerosis in the cholesterol-fed rabbit.

New Zealand White rabbits were injected subcutaneously with either a human dose of bacillus Calmette Guerin (BCG) vaccine (n = 7) or saline (n = 7). A further half dose of BCG or saline was injected after a further 4 weeks. The animals were subsequently fed a 0.25-1% cholesterol diet for 10 weeks, 8 weeks after the first injection. The rabbits were killed and perfusion fixed with 4% paraformaldehyde. The integrated plasma cholesterol levels did not differ significantly between the groups (P > 0.05). Plasma levels of anti-mycobacterial antibodies rose following BCG immunization, reaching a peak after 8 weeks (P < 0.05) compared to basal titers and the control group. BCG immunization was also associated with increased peripheral lymphocyte and monocyte activation, as evidenced by increased surface expression of IL-2 receptor (CD25) (P < 0.02) and MAC-I (CD11b) (P < 0.05), respectively. Significantly more mononuclear cells bound to the aortic endothelium of BCG immunized, cholesterol-fed rabbits (1.93+/-0.77 mononuclear cells/1000 endothelial cells) than to that of saline immunized rabbits (0.08+/-0.08 mononuclear cells/1000 endothelial cells; P < 0.01). The aortic intimal:medial ratio was greater in the BCG immunized rabbits (0.19+/-0.08) than those treated with saline (0.04+/-0.03; P < 0.05). This suggests that BCG immunization enhances peripheral leucocyte activation, aortic monocyte recruitment and atherogenesis in the cholesterol-fed rabbit.

Monophosphoryl lipid A stimulated up-regulation of nitric oxide synthase and nitric oxide release by human monocytes in vitro.

Monophosphoryl lipid A (MPL) is a derivative of lipopolysaccharide (LPS) with reduced toxicity which has been shown to modulate various immune functions in monocytes. We examined whether human monocytes can be stimulated to produce nitric oxide (NO) and its catalytic enzyme nitric oxide synthase (NOS). Monocytes were stimulated with LPS or MPL and both NOS and NO (as nitrite) production were measured. MPL at high doses (> 100 micrograms/ml) stimulated monocytes to release NO that was significantly greater than both the control and LPS-treated monocytes (p < 0.05). NO release by control cells and the LPS treated cells was not significantly different. Both arginase and N-monomethyl arginine (NMLA) inhibited the MPL stimulated release of NO (p < 0.01). MPL significantly increased inducible NOS (iNOS) expression as measured by both fluorescent microscopy and flow cytometry (p < 0.05). Similarly, both soluble NOS (sNOS) and particulate NOS (pNOS) activity were significantly up-regulated by MPL (p < 0.05). Significant correlations were found between pNOS expression and sNOS release (r = 0.72, p < 0.0001) and between 12 h NO release and sNOS production (r = 0.44, p < 0.005). These experiments confirm that human monocytes can be stimulated with MPL to produce NO in vitro and suggest that up-regulation of pNOS does not preclude NO release.

Flow cytometric measurement of non-specific esterase activity.

alpha-Naphthyl acetate esterase (alpha-NAE) is primarily found in mononuclear phagocytes and may be used to distinguish them from other leucocytes. Conventional cytochemical techniques are subjective and may be difficult to interpret, especially with cells which express only low levels of activity. This has caused difficulties in the classification of non-lymphoblastic leukaemias. This paper describes the adaptation of a cytochemical assay for use with the flow cytometer. The alpha-NAE activity of peripheral blood mononuclear cells was examined and found to be associated with the expression of the surface antigen CD14. The reaction could be inhibited by sodium fluoride. A series of human cell lines were also compared for alpha-NAE activity. Distinct differences in staining observed between the cell lines correlated with the number of cell-associated granules observed under the microscope.

Investigations into the mechanisms of carcinogen-induced nuclear enlargement in HeLa S3 cells in vitro.

Carcinogen-induced nuclear enlargement has been reported both in vitro and in vivo, but the mechanism, and whether it is causally related to carcinogenesis, has not yet been established. This study was designed to investigate the role of increased DNA content, such as might occur in polyploidy, in induction of nuclear enlargement. The effects of two genotoxic carcinogens, N-methyl-N-nitrosourea and adriamycin, were compared with the effects induced by diethylstilboestrol, which is arguably a non-genotoxic carcinogen but is known to induce polyploidy. HeLa S3 cells were used as the model system for comparison with previous studies. N-methyl-N-nitrosourea and adriamycin both induced a concentration-related increase in nuclear size 24 to 72 hr after a 30 min pulse-treatment. This was accompanied by an increase in the proportions of cells in the G(2) + M stage of the cell cycle, possibly due to a G(2) block. There was some evidence of polyploidy with adriamycin but not with N-methyl-N-nitrosourea. The distributions of nuclear areas indicated that increases in ploidy contributed to, but did not totally account for, the nuclear enlargement. In contrast, diethylstilboestrol increased the range of nuclear areas and DNA content, to both less than and greater than that of control cells, but only after a prolonged exposure period of 48 hr. These data were consistent with diethylstilboestrol inducing spindle damage. These results demonstrate that carcinogen-induced nuclear enlargement is only partially explained by increased nuclear DNA content, and that certain classes of non-genotoxic carcinogen may produce a completely different pattern to that from genotoxic carcinogens.

An association between Gc (vitamin D-binding protein) alleles and susceptibility to rheumatic fever.

Rheumatic fever is associated with exaggerated activity of B cells with massive production of antibody to the Group A streptococcus. Gc (vitamin D-binding protein) is constitutively expressed on B-cell membranes in association with membrane immunoglobulin, and could be involved in cell activation. We therefore looked for associations between the three major Gc alleles and susceptibility to rheumatic fever in a homogeneous Arab population. Patients with tuberculosis or rheumatoid arthritis and control donors, were studied in parallel. Allele frequencies in the controls, rheumatoid and tuberculosis patients were identical to those found in a previous study of normal Arab donors. However, there was a striking association between Gc2 and rheumatic fever. This allele was twice as common in these patients as in controls (p = 0.0024), and was present in 56.4% of all rheumatic fever patients.

Peripheral blood dendritic cells in persons with AIDS and AIDS related complex: loss of high intensity class II antigen expression and function.

The antigen-specific immune response in HIV sero-positive individuals is depressed or absent. This may be due in part to abnormal co-operation between T lymphocytes and antigen presenting cells (APC). We have isolated from the peripheral blood of healthy heterosexuals and patients with persistent generalized lymphadenopathy (PGL) and AIDS, cells of low density (LDC) with dendritic morphology. These cells are known to be potent APC. The expression of two cell surface antigens on these cells, namely 63D3 (a monocyte related antigen) and Class II antigens was examined. LDC from controls and patients with benign, non-progressive PGL (type A) were found to show biphasic expression of Class II antigens. By contrast, the high intensity Class II expression seen on a small proportion of 63D3 negative cells from controls and patients with PGL type A was absent in patients with PGL type B (showing subtle signs of progressive immunodeficiency) and AIDS. The loss of this population of dendritic cells was reflected in the absence of stimulator activity in autologous and heterologous mixed lymphocyte reactions. Thus, it is possible that the loss of these dendritic cells may contribute to the profound immunological abnormalities seen in AIDS.

Association of different allelic forms of group specific component with susceptibility to and clinical manifestation of human immunodeficiency virus infection.

The distribution of phenotypes of the group specific component (Gc) was examined in 203 homosexuals at risk of infection or infected by the human immunodeficiency virus and compared with that in 50 randomly selected homosexuals and 122 healthy male heterosexual seronegative controls. 30.2% of patients with the acquired immunodeficiency syndrome (AIDS) were homozygous for Gc 1 fast (Gc 1f) compared with 0.8% of controls (p less than 0.0001); patients with other clinical manifestations of HIV infection were also more likely than controls to have Gc 1f. By contrast, seronegative symptomless homosexual contacts of AIDS patients (AH-p) lacked this phenotype but were more likely than controls to be homozygous for Gc 2 (25% vs 9%, p less than 0.05). AIDS patients lacked the homozygous Gc 2 phenotype altogether. A chi 2 trend test showed that progression to AIDS had a strong positive association with the Gc 1f allele (p less than 0.0001) and a negative one with Gc 2 (p less than 0.05). It is proposed that Gc may be involved in viral entry into host cells, the ease of which varies with different allelic forms of Gc, according to their sialic acid content.

Atopic manifestations in the acquired immune deficiency syndrome: response to recombinant interferon gamma.

Six patients with the acquired immune deficiency syndrome (AIDS) had exacerbations or recurrences of previously quiescent atopic disease when they developed immunodeficiency. Four developed a different atopic illness from that suffered previously. Atopic symptoms developed within three months after the patients developed AIDS or during prodromal illness. Two of the patients were treated with recombinant interferon gamma: both showed a striking improvement in symptoms and cellular immunity. These results indicate that cellular immunity, through interferon gamma, may have a role in regulating atopic disease.

Microbicidal activity of monocyte derived macrophages in AIDS and related disorders.

We have examined the ability of monocyte-derived macrophages from patients with AIDS and other HIV-related disorders to kill the intracellular pathogen Toxoplasma gondii. We have also examined the capacity of peripheral blood mononuclear cells from these patients to produce macrophage-activating and other lymphokines. The capacity to produce interleukin 2 and gamma interferon decreases from controls through asymptomatic seropositive subjects and lymphadenopathy groups A (benign) and B (prodromal) to AIDS. The decrease did not correlate precisely with the decrease in CD4+ cells in these patients. Monocyte-derived macrophages from asymptomatic HIV-infected subjects and lymphadenopathy patients showed a decreased ability to kill T. gondii after activation with recombinant gamma interferon; paradoxically, this was most striking for PGL group A. The defect was largely overcome by using Concanavalin A stimulated autologous supernatants. It was notable that macrophages from AIDS patients showed normal killing with recombinant gamma interferon, but that the supernatants from AIDS patients had reduced activity with normal macrophages. These studies confirm that functional defects of both lymphocytes and macrophages are found in HIV-infected subjects; they serve to emphasize the heterogeneity of the clinical and biological responses to this retrovirus, responses which have important implications in the pathogenesis and treatment of the immunodeficiency.

Lymphocytes bearing Fc gamma receptors in rheumatoid arthritis. IV. Increased numbers and activation of Facb-R+ cells after immunisation of healthy individuals.

Mononuclear cells expressing Fc gamma receptors that form Facb rosettes are increased in the peripheral blood of patients with rheumatoid arthritis compared with controls. Healthy individuals with a positive skin response to tuberculin showed a marked increase in numbers of circulating Facb-R+ cells three days after challenge, returning to baseline after seven days. No response was observed in subjects showing a negative skin test. A similar increase in Facb-R+ cell numbers was measured after intramuscular injection of another specific antigen, tetanus toxoid. In addition to this enhancement of Facb-R+ cell numbers, evidence has been obtained that these cells are in an activated state postimmunisation as judged by acquisition of low density and increased expression of class II MHC antigens. Apparently identical changes in Facb-R+ cell numbers and activation may be induced in vitro either by culturing sensitised mononuclear cells with specific antigen for three days or by an overnight incubation of normal cells with gamma-interferon (gamma-IFN). By analogy, therefore, the increased numbers of Facb-R+ cells in patients with rheumatoid arthritis are probably induced by gamma-interferon generated as part of an antigen driven immune response. In this context it is interesting that patients with Felty's syndrome, in whom neutropenia increases susceptibility to infections leading to the possibility of further stimulation of the immune system by micro-organisms, have particularly high levels of circulating Facb-R+ cells.

Lymphocytes bearing Fc gamma receptors in rheumatoid arthritis. II. Phenotypic characterisation of mononuclear cells forming Facb rosettes in RA.

We have previously reported an increased proportion of Facb-rosette forming cells in the peripheral blood of patients with rheumatoid arthritis in comparison with healthy controls. The present study investigates the surface phenotype of these cells by means of monoclonal antibodies and a variety of rosetting and lymphocyte fractionation techniques. Facb-R+ cells were found to lack surface markers characteristic of T and B lymphocytes. Studies with monoclonal reagents showed a positive reaction with OKIa1, OKM1, and another monocyte-specific antibody. Fac-R+ cells were recognised by anti-HLA-DR reagents but did not bind the monoclonal antibody 17.15 that recognises a determinant on HLA-DR antigens expressed by lymphocytes but not monocytes. These results show that Facb-R+ cells share certain surface characteristics with monocytes, though they are not phagocytic. These observations are consistent with an accessory role for Facb-R+ cells in the immune response.

Lymphocytes bearing Fc gamma receptors in rheumatoid arthritis. III. Immunoregulatory function associated with Facb rosette-forming cells.

A subpopulation of mononuclear cells (PBMNC) that expresses Fc receptors with specificity for the C gamma 2 region of IgG may be detected by rosette formation with calf erythrocytes coated with the Facb fragment of rabbit IgG. These Facb-R+ cells are found in increased numbers in the peripheral blood of patients with rheumatoid arthritis (RA). Studies have been carried out to identify the functional properties of these cells in healthy and rheumatoid subjects. Facb-R+ cells were shown to lack both natural killer and antibody-dependent cytotoxic activity. Depletion of Facb-R+ cells from both healthy and rheumatoid PBMNC resulted in a marked suppression of pokeweed mitogen (PWM) stimulated IgG synthesis but had no effect on T cell proliferation induced by phytohaemagglutinin, concanavalin A, or PWM. The addition of Facb fragments to PBMNC cultures also caused inhibition of PWM-driven IgG production. In this assay rheumatoid PBMNC were significantly less sensitive to Facb-mediated suppression than healthy control cells. Our results suggest that Facb-R+ cells are involved in the antibody-mediated feedback regulation of immunoglobulin synthesis and that this mechanism is impaired in patients with RA.

Facb rosette-forming cells in mice: studies on their functional significance.

Lymphocytes bearing receptors for the Facb fragment of IgG have been shown previously to be elevated in the peripheral blood of patients with rheumatoid arthritis. The generation of these cells and their possible functional role in immune regulation have been investigated in mice. Facb rosette-forming (Facb-R+) lymphocytes were found to be elevated in the spleens of mice mounting a secondary plaque-forming cell (PFC) response to sheep erythrocytes but not during the primary response. Splenic Facb-R+ lymphocytes were also elevated when a cross-reacting antigen (goat erythrocytes) was used for the secondary immunization but not when a non-cross-reacting antigen (chicken erythrocytes) was used. Both primary and secondary immunization with bacterial lipopolysaccharide resulted in elevation of splenic Facb-R+ lymphocytes. Administration of antigen-specific Facb fragment in conjunction with antigen (calf erythrocytes) produced a suppression of the secondary PFC response. However, F(ab')2 fragments produced no such effect. This suppressive effect was shown to be antigen-specific since administration of Facb fragment of anti-calf erythrocyte IgG had no suppressive effect on the secondary PFC response to sheep erythrocytes. No change in splenic Facb-R+ lymphocytes was observed during delayed hypersensitivity responses to either sheep erythrocytes or the contact-sensitizing agent oxazolone. These results indicate that Facb-R+ lymphocytes are generated during secondary humoral responses but not cell-mediated immune responses, and suggest that these cells may exert a suppressive influence on antibody production. These findings are discussed in relation to the occurrence of these cells in patients with rheumatoid arthritis.