Intracellular cytokine profile in T-cell subsets of multiple sclerosis patients: different features in primary progressive disease.

OBJECTIVE: To evaluate the expression of cytokines in both CD4+ and CD8+ T cells derived from peripheral blood of untreated multiple sclerosis (MS) patients with either relapsing-remitting (RR), secondary progressive (SP) or primary progressive (PP) MS and healthy controls (HC). BACKGROUND: MS is an immune-mediated disease and cytokines hove been hypothesized to contribute significantly to disease progression. Compared to the relapse-onset (RR, SP) form of the disease, PPMS patients have different clinical, immunological and pathological features. Surprisingly, the ability of their circulating T cells to produce immunoregulatory cytokines has not been extensively studied so far. METHODS: Seventy-two MS patients (24 RR, 26 SP, 22 PP) and 34 HC were studied. Stimulated peripheral blood derived CD4+ and CD8+ T MS patients express significantly more CD4+ and CD8+ T cells were analyzed for IFN-gamma, IL-2, TNF-alpha, IL-4, IL-10 and IL-13 production. RESULTS: cells producing IFN-gamma compared to HC. Compared to the other forms of the disease, PPMS patients display a significant decrease in CD4+ T cells producing IL-2, IL-13 and TNF-alpha and a significant increase in CD8+ T cells producing IL-4 and IL-10. CONCLUSIONS: The data presented here demonstrate that patients with PPMS express less pro- and more anti-inflammatory cytokine producing T cells compared to the relapse-onset form of the disease, confirming the view on PPMS as a distinct disease entity.

Aberrant expression and reverse signalling of CD70 on malignant B cells.

In normal lymphoid tissues the tumour necrosis factor-receptor family member CD27 and its ligand CD70 have a restricted expression pattern. Previously, we reported that expression of CD27 is deregulated in B-cell leukaemias and lymphomas. Here we show that, although infrequently expressed by normal human B cells in vivo, CD70 is found on 50% of B-CLLs, 33% of follicle centre lymphomas, 71% of large B-cell lymphomas, and 25% of mantle cell lymphomas. Interestingly, in the majority of leukaemias and lymphomas examined, CD70 was found to have a capped appearance, a feature that coincided with co-expression of CD27. Functional analysis showed that a subset of B-CLLs could proliferate vigorously in response to CD70 mAb but not to CD27 mAb. This response was synergistically enhanced by ligation of CD40 but inhibited by the presence of IL-4. Additional experiments indicated that the proliferative response was due to an agonistic signal delivered via CD70, rather than blocking of negative signalling by CD27. Thus, next to its role as ligand, in a subset of malignant B cells CD70 can operate as receptor and as such might contribute to progression of these B-cell malignancies.

Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells.

To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1beta-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.

A dual role for both CD40-ligand and TNF-alpha in controlling human B cell death.

Members of the TNF-R family are instrumental in controlling lymphoid cell death and survival. A major role in the regulation of murine and human B cell survival and differentiation has been attributed to CD40/CD40-ligand (CD40-L) interactions, but recent in vitro and in vivo data implicate that other receptor-ligand pairs might also be involved. We have used the human Burkitt lymphoma cell line Ramos as a model system to identify additional TNF-R/TNF family members that are implicated in the regulation of B cell apoptosis. Ligation of B cell receptor (BCR) with anti-IgM mAb for 48 h induced apoptosis in approximately 68% of the Ramos B cells. Interestingly, not only CD40 mAb but also rTNF-alpha could efficiently inhibit BCR-induced B cell death. In addition, activated T cells also prevented BCR-triggered apoptosis, and this effect was inhibited completely by a combination of blocking Abs against CD40-L and TNF-alpha. In contrast to the strong effect of BCR ligation, APO-1 mAb induced apoptosis in only +/- 18% of the Ramos cells after 48 h. Noticeably, addition of CD40 mAb or rTNF-alpha increased the percentage of cells (+/- 46%) undergoing apoptosis, which correlated with an increase of Fas/APO-1 membrane expression induced by CD40 or TNF-R ligation. Taken together, we show that CD40/CD40-L and TNF-R/TNF-alpha interactions not only postpone or prevent B cell death, but are also involved in sensitizing B cells for Fas-ligand (Fas-L)-dependent death.