The amplifier role of T cells in the human in vitro B cell response to type 4 pneumococcal polysaccharide.

The human B cell response to T cell independent type 2 antigens is regulated by thymus-derived lymphocytes. We analyzed the role of T cells in the in vitro antibody response to type 4 pneumococcal polysaccharide (PS4). We here show that the amplifying effect of T cells, which has previously been shown to be radioresistant and confined to T cell preparations enriched for CD4+ cells, is MHC non-restricted as demonstrated in cultures carried out in the presence of allogeneic T cells. Also, T cell clones derived from non-related donors are able to enhance the B cell response to PS4. All TCR alpha beta +, CD 4+ T cell clones, but none of the TCR alpha beta +, CD 8+ T cell clones tested, enhanced the B cell response to PS4. Furthermore, 3 out of 6 TCR gamma delta+ T cell clones were capable of enhancing the anti-PS4 B cell response. Experiments using recombinant lymphokines and glutaraldehyde-fixed T cells indicated that both lymphokines and T-B cell interactions are required for an optimal antibody response to PS4.

Role of CR2 in the human adult and neonatal in vitro antibody response to type 4 pneumococcal polysaccharide.

A number of studies have indicated that the complement receptor type 2 (CR2), which is the receptor for C3d, a degradation fragment of the complement component C3, regulates B lymphocyte activation and growth. Early reports have described that C3 regulates T cell-dependent (TD) antibody responses. The involvement of CR2 in the antibody response to T cell-independent type 2(TI-2) antigens was investigated because neonatal B cells, which are unresponsive to TI-2 antigens both in vivo and in vitro, express a significantly decreased level of CR2 as compared to B cells of adult donors. We utilized type 4 pneumococcal polysaccharide (PS4) as a model TI-2 antigen. In order to study the relationship between CR2 and the response to PS4, B cells were costimulated with PS4 and monoclonal antibodies (MAb) to CR2. HB5 and OKB7 anti-CR2 monoclonal antibodies enhanced the in vitro response of adult B cells to PS4, as measured in a PS4-specific spot-forming cell assay. Neonatal B cells could only be induced to respond to PS4 using high concentrations of OKB7 anti-CR2 MAb. The 8-mercaptoguanosine (8MGuo), an agent that can overcome the in vitro unresponsiveness to PS4 of neonatal B cells, increased CR2 expression on adult and neonatal B cells. Furthermore, 8MGuo synergizes strongly with anti-CR2 antibodies in augmenting the anti-PS4 antibody response. Data presented in this report provide evidence of CR2 involvement in the antibody response to PS4 and that the neonatal B cell unresponsiveness to TI-2 antigens may be due to the decreased expression of CR2.

The human in vitro anti-type 4 pneumococcal polysaccharide antibody response is regulated by suppressor T cells.

In order to study the regulation of the human B-cell response to T-cell independent type 2 (TI-2) antigens, we analysed the role of T cells in the in vitro antibody response to type 4 pneumococcal polysaccharide (PS4). We found that T cells can positively regulate the in vitro antibody response to PS4 when they are added into an in vitro B-cell culture system. In addition we demonstrated that T cells exert a negatively regulating activity. We found that T cells, when cultured for 24 h in the presence of high concentrations of PS4, can suppress the anti-PS4 antibody response. This down-regulation of the anti-PS4 B-cell response is shown to be antigen specific and MHC-restricted. Furthermore, PS4-specific T-cell mediated suppression appears to be radioresistant and confined to the T-cell preparations enriched for CD8+ cells. The results show that analogous to results obtained in mice, human T cells are able to exert a regulatory control of the antibody response to TI-2 antigens.

Expression of deoxyadenosine and deoxyguanosine toxicity at different stages of lymphocyte activation.

We have previously shown that deoxyguanosine (dGuo) is toxic to normal T and B lymphocytes, an effect mediated by intracellular accumulation of guanine ribonucleotides. In order to define the cellular processes that are sensitive to guanosine triphosphate (GTP) we have performed studies in which the effects of dGuo on normal T cells are compared with those of deoxyadenosine (dAdo) on adenosine deaminase (ADA)-deficient T cells. Kinetic studies show that dAdo exerts its toxic effects on processes that precede the onset of DNA synthesis, like interleukin 2 receptor expression, whereas dGuo added as late as 24-48 h after initiation of the culture still inhibits mitogen-induced proliferation. It can thus be concluded that dGuo toxicity as mediated through guanine ribonucleotides is manifested relatively late during the process of T-cell activation, whereas dAdo acts early in T-cell activation by a mechanism that cannot be explained by inhibition of ribonucleotide reductase.

Bactericidal/permeability-increasing protein (BPI) inhibits angiogenesis via induction of apoptosis in vascular endothelial cells.

Bactericidal/permeability-increasing protein (BPI) has been known for some time to function in killing bacteria and in neutralizing the effects of bacterial endotoxin lipopolysaccharide. In the present study, BPI is found to be a novel endogenous inhibitor of angiogenesis. Within the sub-muM range, BPI shows a concentration-dependent inhibition of endothelial cell (EC) proliferation that is mediated by cell detachment and subsequent induction of apoptosis. As measured by flow cytometric analysis of the percentage of subdiploid cells, apoptosis induction was half-maximal at about 250 nmol/L BPI. Apoptosis was confirmed by quantification of cells with nuclear fragmentation. Apoptosis was found to be EC specific. In an in vitro collagen gel-based angiogenesis assay, BPI at 1.8 micromol/L inhibited tube formation by 81% after only 24 hours. Evidence for in vivo inhibition of angiogenesis was obtained, using the chorioallantoic membrane assay in which BPI was seen to be significantly effective at concentrations as low as 180 nmol/L. This newly discovered function of BPI might provide a possible therapeutic modality for the treatment of various pathologic disorders that depend on angiogenesis.

Different pathways for deoxyguanosine toxicity in T-lymphocytes of various developmental stages.

The basis of the selective cellular immunodeficiency which occurs in patients with purine nucleoside phosphorylase (PNP) deficiency still is not completely understood. We studied the mechanism of deoxyguanosine (dGuo) toxicity in proliferating lymphoid T-cells of different maturation stage, i.e. in T-cells of adult peripheral blood and cord blood and in CD3+ and CD3- subfractions of thymocytes. The mitogen-induced proliferation of T-cells from peripheral blood and cord blood and of CD3+ and CD3- subfractions of thymocytes. The mitogen-induced proliferation of T-cells from peripheral blood and cord blood and of CD3+ thymocytes, as well as the spontaneous proliferation of CD3- thymocytes, are inhibited by dGuo. CD3+ and CD3- thymocytes are significantly more sensitive to dGuo than T-cells from peripheral blood or cord blood. Among the thymocyte subfractions CD3- thymocytes appeared to be extremely sensitive. In all cell types studied, inhibition of proliferation is accompanied by intracellular increases in both guanosine triphosphate (GTP) and deoxyguanosine triphosphate (dGTP) concentrations. By use of the PNP inhibitor 8-aminoguanosine, or the metabolites hypoxanthine or deoxycytidine, the metabolism of dGuo could be selectively directed to the formation of GTP or to dGTP. Based on the pattern of rescue from dGuo intoxication under these different metabolic conditions we conclude that in CD3- thymocytes dGuo toxicity is mediated by dGTP. In all other cell types studied GTP mediates dGuo intoxication. Altogether the results show that during the maturation from immature thymocytes to mature peripheral blood T-cells a shift occurs in the pattern of dGuo toxicity since dGuo toxicity in the former is primarily caused via the dCyd kinase pathway, and in the latter mainly the degradation route is involved. Since in PNP deficiency mature T-cells do occur in the peripheral blood, we must conclude that some cells escape the stage of T-cell maturation in the thymus which is extremely sensitive to dGuo. Furthermore, the results imply that as far as T-cell development in the normal thymus is concerned, survival and death of cells might be regulated by local (deoxy) nucleoside availability.

Autoreactivity to human heat-shock protein 60 predicts disease remission in oligoarticular juvenile rheumatoid arthritis.

OBJECTIVE: To determine whether T lymphocyte reactivity to endogenous human hsp60 plays a regulatory role in the course of oligoarticular juvenile rheumatoid arthritis (JRA). METHODS: A prospective, longitudinal study of T cell reactivity to HSP in 15 patients with newly diagnosed HLA-B27 negative oligoarticular JRA was performed. Results were compared with those in a group of 20 patients with newly diagnosed polyarticular or systemic JRA or with acute arthritis caused by other systemic diseases or viral infections, as well as with those in a group of 9 healthy control subjects. RESULTS: In 86% of the patients with oligoarticular JRA (13 of 15), significant T lymphocyte proliferative responses to hsp60 were found in peripheral blood mononuclear cells and/or synovial fluid mononuclear cells within 3 months after the onset of arthritis. Only 5% of the patients in the rheumatologic disease control group (1 of 20) showed such positivity. All patients with oligoarticular JRA and positive responses to human hsp60 developed a remission of their disease within 12 weeks. During this period of remission, blood samples were taken from 8 patients and showed significantly lower and even negative responses to hsp60, compared with active disease, when all 8 patients had good responses. CONCLUSION: The results show that significant proliferative responses to human hsp60 can be found early in the course of oligoarticular JRA. Furthermore, these responses correlate with disease activity in such a manner that T cell reactivity to human hsp60 seems to be associated with disease remission.