Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-alpha.

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-alpha upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of beta-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-alpha release peaked after only 6 h. Anti-TNF-alpha mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-alpha. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.

Value of prothrombin fragment 1.2 (F 1.2) in the diagnosis of stroke in young patients with antiphospholipid antibodies.

The presence in the serum of antiphospholipid antibodies (aPL) is associated with venous and arterial thrombosis. This observation has led to the search for these antibodies in young patients with ischemic neurologic syndromes. However, 1% to 5% of healthy people may be found to have circulating aPL without necessarily being at increased risk of thromboembolism. Thus, the finding of APLA in a patient with cerebral ischemia does not necessarily provide an explanation for the etiology of the clinical syndrome. The aim of this study was to determine whether the presence of aPL in young patients with stroke or transient ischemic attacks represents a possible cause of hypercoagulability as defined by ongoing thrombin formation with resultant elevation of prothrombin fragment 1.2 (F1.2) levels. This was a retrospective, case-control study involving 57 subjects. Twenty-seven patients had a recent cerebrovascular ischemic event--either TIA or a stroke. Fifteen were positive for aPL, and 12 were aPL-negative. Thirty subjects, matched for age and sex with no history of cerebrovascular disease, served as controls. Of this group, 20 were aPL-positive and 10 were aPL-negative. Causes of hypercoagulability other than aPL were excluded by laboratory testing. A positive test for aPL was repeated after a 6-week interval and two positive tests were required for a patient to be regarded as being aPL-positive. Levels of F1.2 were measured by an ELISA technique. There was a significant difference (p < 0.05) in the mean F1.2 levels between the aPL-positive group with a history of cerebrovascular disease (mean F1.2 = 2.3733) and each of the other study groups. There was no statistically significant difference between any of the other study groups. Our findings suggest that F1.2 levels are elevated in young patients with cerebrovascular syndromes who have aPL and in whom other causes of hypercoagulability and atherosclerotic vascular disease are absent. Elevated F1.2 in these patients may be a potential marker of the hypercoagulable state associated with aPL.

Activated T lymphocytes induce degranulation and cytokine production by human mast cells following cell-to-cell contact.

Activated mast cells reside in close apposition to T cells in some inflammatory processes. In this study, we analyzed whether this close physical proximity affects human mast cell degranulation and cytokine release. Thus HMC-1 human mast cells or primary bone marrow-derived human mast cells were cocultured with activated and with resting T cells. Mast cells cocultured with activated T cells released histamine and beta-hexosaminidase and produced tumor necrosis factor alpha (TNF-alpha), an effect that peaked at 20 h. Kinetics of histamine release paralleled the formation of heterotypic aggregates. Separation of the two cell populations with a porous membrane prevented mediator release and TNF-alpha production. Addition of the PI3-kinase inhibitor, wortmannin, inhibited the heterotypic adhesion-associated degranulation but not TNF-alpha production. These data thus indicate a novel pathway through which human mast cells are activated to both release granule-associated mediators and to produce cytokines in association with heterotypic adhesion to activated human T cells.

Inhibitory effects of low molecular weight heparin on mediator release by mast cells: preferential inhibition of cytokine production and mast cell-dependent cutaneous inflammation.

There has been substantial evidence that suggests that heparin may modulate various aspects of immune function and inflammation in addition to its well known anticoagulant activity. In this regard heparin was found to suppress cell-mediated immune responses or asthmatic reactions to allergen challenge. In the present study we analyse the effects of low molecular weight heparin (LMWH) on mast cell degranulation and cytokine production in vitro and on the elicitation of IgE-mediated mast cell-dependent late cutaneous allergic inflammation in vivo. We have established that LMWH preferentially inhibited tumour necrosis factor-alpha (TNF-alpha) and IL-4 production without having any significant effect on mast cell degranulation. These effects have been observed in mast cells derived from three different origins that were activated by either immunological or non-immunological stimuli. We have shown that there is inhibition of TNF-alpha production (and not neutralization of activity), as elimination of the drug after a short preincubation and addition of LMWH to rTNF-alpha had no effect on TNF-alpha-mediated cytotoxic activity. These results were also confirmed by ELISA. In vivo, s.c. injection of the LMWH inhibited the leucocyte infiltration associated with the late cutaneous response which followed passive cutaneous anaphylaxis (PCA) reaction, without affecting mast cell numbers or degranulation. These data suggest that LMWH may have an inhibitory role in mast cell-mediated allergic inflammation, and thus might be considered as a possible therapeutic modality.

Interleukin-6 and interleukin-6 receptor secretion by chronic lymphatic leukaemia and normal B lymphocytes: effect of PMA and PWM.

Interleukin-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R) were detected in supernatants of cultures of B chronic lymphatic leukaemia (CLL) lymphocytes. Phorbol-12-myristate 13 acetate (PMA) caused a decrease in the levels of IL-6 in 14 out of 16 cultures and an increase in levels of sIL6R in all 15 cases. The effect of pokeweed mitogen (PWM) was variable and not significant. The levels of IL-6 were below the detection limit (60 pg/ml) in sera of 13 CLL patients whereas sIL-6R was detected (13 ng/ml to 97 ng/ml) in the 13 sera. IL6 was not detected in cultures of unstimulated or stimulated with PMA or PWM normal human B cells. Levels of sIL-6R were minimal in cultures of normal B lymphocytes and were increased in PMA stimulated cultures. The results are consistent with the view that B-CLL cells produce spontaneously IL-6 which could act in an autocrine fashion to cause shedding of surface IL-6R and account for the correlation found between serum levels of sIL-6R and B-CLL lymphocyte numbers. The fall in levels of IL-6 in PMA stimulated CLL cultures might express masking or degradation of IL-6 after combination with the receptor.

Secretion of suppressor factors by EBV infected B cell lines.

The infection of human peripheral blood B cells with Epstein-Barr Virus (EBV), induced the production of suppressor factor(s) which were released into the supernatant of the B-cell cultures. The induction of suppressive activity was independent of T-cell presence. The suppression was exhibited both against T-cell activity (MLR and mitogenic stimulation) as well as against B-cell mitogenic stimulation of human or murine B lymphocytes. The suppressive factor(s) was of a low molecular weight (equal or less than 5,000), resistant to trypsin and heating at 80 degrees C and its activity was partially inhibited by neuraminidase treatment. These findings indicate that the suppressive factor(s) is not correlated to immunoglobulin production, is not apparently of a protein nature, and might be of ganglioside or siaylated glycoprotein structure. Our present findings suggest that, in addition to T cells, B cells might also play an immunoregulatory role in the expression of immune response potential.

Inhibition by uraemic sera of CD4+ T-cell adhesion to extracellular matrix components.

BACKGROUND: T-cell-mediated immune responses are impaired in patients with chronic renal failure. The migration, proliferation, differentiation, biological functioning, and interaction with other T cells are mediated by cell surface adhesion proteins, which include integrins. METHODS: To elucidate how uraemia can impair T-cell-mediated responses in vivo, the effects of sera from uraemic patients on T-cell proliferation and adhesion to extracellular matrix (ECM) components were examined. RESULTS: Preincubation of human CD4+ T cells with sera from undialysed and dialysed (haemodialysis or peritoneal dialysis) uraemic patients inhibited the capacity of the cells to be stimulated by phytohaemmagglutinin and by anti-CD3 monoclonal antibody plus immobilized fibronectin (FN). Sera from uraemic and dialysed patients, but not from healthy individuals, inhibited significantly, and in a dose-dependent fashion, human CD4+ T cell adhesion to immobilized FN and laminin (LN). The degree of inhibition of adhesion was similar whether the sera were continuously present, even during the adhesion assay, or removed by washing. The adhesion inhibiting capacity of the uraemic sera was not due to modification of the expression of beta 1 integrins on the surfaces of the T cells. CONCLUSIONS: These results suggest that uraemia can impair the proliferative capacity and adhesion of immune cells, and thus may affect normal immune processes and contribute to the overall immune deficiency observed in patients with renal failure.

Immunosuppression in vitro and in vivo by supernatants from murine lymphoma cell lines.

Supernatants from four murine B and one T-cell lymphoma cultures were tested for their suppressive effect on allogeneic response in vitro (MLR), on their effect on mitogenic response to ConA and LPS and on their effect in vivo on allogeneic stimulation of BALB/c mice by C57BL/6J spleen cells. Two out of four B-cell supernatants (WEHI-231 and 24-666) were also tested for their effect on induction of IL-2 like activity by ConA in BALB/c spleen cells and on the effect of their injection on subsequent allogeneic response of spleen cells from BALB/c mice immunized against C57BL/6J spleen cells. The supernatants from all lymphoma lines inhibited MLR but only supernatants from the B-cell lymphoma lines also inhibited mitogenic stimulation in vitro and allogeneic immunization. The two B-cell supernatants tested also inhibited induction of IL-2 like activity and decreased the ability of spleen cells from the injected mice to respond to allogeneic stimulation. Partial characterization of supernatants from WEHI-231 and 24-666 lymphoma cultures revealed the non-protein nature of the suppressor factor(s), indicated a molecular weight of less than 5 Kd and suggested a ganglioside or a sialyated glycoproteinic nature.

T lymphocytes of rheumatoid arthritis patients show augmented reactivity to a fraction of mycobacteria cross-reactive with cartilage.

An acetone-precipitable fraction of Mycobacterium tuberculosis cross-reacts with human cartilage. Immune responses to this antigen were assessed in 34 patients with rheumatoid arthritis, 16 patients with degenerative joint disease, and 15 healthy controls. The RA patients differed from the other two groups in having more pronounced T lymphocyte responses to the antigen; their serum antibody levels were not higher. The responses of RA patients varied with duration of disease. In the first year (7 patients) T lymphocyte reactivity was increased in the synovial exudates of affected joints but not in peripheral blood, whereas the 19 with disease of 1-10 years' duration showed high reactivity in peripheral blood; in the 8 with disease for more than 10 years, lymphocyte reactivity did not differ from that in the patients with degenerative joint disease or the healthy controls. The observation that the three groups did not differ in their responses to streptococci and a T-cell mitogen indicates that reactivity of the RA patients to the mycobacterial fraction was specific. These results raise the possibility that bacterial antigens cross-reactive with cartilage proteoglycans may be relevant to the pathogenesis of RA.

In vitro effects of 4-hydroperoxycyclophosphamide on concanavalin A-induced human suppressor T cells.

Treatment in vitro of human peripheral blood lymphocytes (PBL) with ConA induced the generation of suppressor cells which inhibited T cell blastogenic response to ConA and of allogeneic response in the mixed lymphocyte reaction (MLR). Treatment of PBL with 4-hydroperoxycyclophosphamide (4-HPCy) before incubation with ConA markedly decreased the generation of suppressor cells by ConA. The effect of 4-HPCy on generation of suppressor cells was more pronounced in the test of ConA stimulation than in the MLR. Treatment with 4-HPCy had no effect on suppressor cells already induced as shown by incubation of PBL with 4-HPCy after incubation with ConA.

The prevalence of antibodies to histones induced by procainamide in old people, in cancer patients, and in rheumatoid-like disease.

Antibodies to histones were found to be most commonly responsible for the positive antinuclear antibody (ANA) test in asymptomatic patients treated with procainamide, in old people, in patients with neoplastic diseases, and in young women affected with a rheumatoid-like disease. Only in a very few patients were antibodies to dDNA and nucleoproteins demonstrated. Antibodies to nDNA were not found. The antibodies to histones were demonstrated by two methods: absorption of ANA-positive sera with a histone solution and subsequent performance of an ANA test; and acid elution of histones from thyroid sections followed by histone reconstitution.

Autologous mixed lymphocyte reaction in man: further characterization of responding cells.

Experiments were performed to determine the type of cells undergoing thymidine incorporation in 7-day autologous mixed lymphocyte reactions (AMLR). Peripheral blood mononuclear cells (PBM) were separated into B cells, T cells, B + Null cells and T + Null cell-enriched populations. Cells were cultured in various combinations. Monocytes were removed to determine their influence on AMLR. The main thymidine-incorporating cells in cultures were shown to be Null cells and to a lesser extent T cells. Monocytes were found to have a more pronounced suppressor effect on stimulation of T cells by non-T cell populations in younger individuals than in the elderly. Whether Null cells undergo a spontaneous blastogenesis in culture or are stimulated by T or B cells, could not be answered by the present investigation.

Monocyte-mediated regulation of cellular immunity in humans: loss of suppressor activity with ageing.

The effect of ageing on cellular immunity in humans was investigated. Human T cell cytotoxicity (CMC) measured by an in vitro xenogeneic assay was found previously to be depressed in individuals greater than 60 years old (group 2) compared to individuals less than 50 years old (group 1). Removal of plastic-adherent cells prior to sensitization in the xenogeneic CMC assay of human peripheral blood mononuclear cells (HPBM) resulted in a significantly higher rise (P less than 0.001, Wilcoxon rank test) in CMC activity in group 1 compared to group 2. Replacement of plastic-adherent cells (greater than 35% monocytes) to HPBM depleted of monocytes returned the CMC activity to the level observed with unseparated HPBM. Separation of HPBM on Percoll density gradients into monocyte-enriched (less than 75%) and monocyte-depleted (less than 3%) fractions indicated that monocytes were responsible for suppressing CMC in group 1. These results are consistent with the hypothesis that monocyte suppressor function for CMC response declines in ageing humans. When indomethacin (1 microgram/ml) was added to HPBM the Con A- and PHA-induced DNA synthetic response rose in groups 1 and 2. Indomethacin induced a significantly larger (P less than 0.01) rise in suboptimal PHA mitogenesis in group 1 compared to group 2. Individuals whose CMC response rose following adherent cell depletion were examined for the effect of indomethacin on the CMC response of their HPBM. In nine of 13 cases, addition of indomethacin also resulted in increased CMC activity.

Cell-mediated immune response to bacterial products in human tonsils and peripheral blood lymphocytes.

Lymphoproliferative responses of tonsillar tissue lymphocytes and peripheral blood lymphocytes to phytohemagglutinin and specific bacterial product antigens were studied in children undergoing tonsillectomy and adenoidectomy. Tonsillar tissue lymphocytes responded to optimal concentrations of phytohemagglutinin. Varidase, and streptolysin-O in a manner similar to peripheral blood lymphocytes. Higher base-line mitogenic activity in tonsillar lymphocytes was frequently associated with the presence of Staphylococcus aureus in the tonsils. Tonsillar tissue lymphocytes from 23% of the subjects with the highest base-line mitogenic activity manifested a decreased response to in vitro stimulation with mitogens or antigens. In subjects with such preactivated tonsillar lymphocytes, the proliferative responsiveness of blood lymphocytes to mitogen and antigens was markedly increased after tonsillectomy and adenoidectomy. These observations suggest the existence of in vitro correlates of cellular immunity to bacterial products in the mucosal surfaces. In addition, it is proposed that tonsils may possess immunosuppressive activity for peripheral blood lymphocytes, which may be related to local tonsillar infections.