A predominant Th1 polarization is present in synovial fluid of end-stage osteoarthritic knee joints: analysis of peripheral blood, synovial fluid and synovial membrane.

Thorough understanding of the complex pathophysiology of osteoarthritis (OA) is necessary in order to open new avenues for treatment. The aim of this study was to characterize the CD4+ T cell population and evaluate their activation and polarization status in OA joints. Fifty-five patients with end-stage knee OA (Kellgren-Lawrence grades III-IV) who underwent surgery for total knee arthroplasty (TKA) were enrolled into this study. Matched samples of synovial membrane (SM), synovial fluid (SF) and peripheral blood (PB) were analysed for CD3+ CD4+ CD8- T cell subsets [T helper type 1 (Th1), Th2, Th17, regulatory T cells] and activation status (CD25, CD69, CD45RO, CD45RA, CD62L) by flow cytometry. Subset-specific cytokines were analysed by cytometric bead array (CBA). SM and SF samples showed a distinct infiltration pattern of CD4+ T cells. In comparison to PB, a higher amount of joint-derived T cells was polarized into CD3+ CD4+ CD8- T cell subsets, with the most significant increase for proinflammatory Th1 cells in SF. CBA analysis revealed significantly increased immunomodulating cytokines [interferon (IFN)-γ, interleukin (IL)-2 and IL-10] in SF compared to PB. Whereas in PB only a small proportion of CD4+ T cells were activated, the majority of joint-derived CD4+ T cells can be characterized as activated effector memory cells (CD69+ CD45RO+ CD62L- ). End-stage OA knees are characterized by an increased CD4+ T cell polarization towards activated Th1 cells and cytokine secretion compared to PB. This local inflammation may contribute to disease aggravation and eventually perpetuate the disease process.

[Regulatory B cells and their role in maintaining peripheral tolerance]. / Regulatorische B-Zellen und ihre funktion im erhalt der peripheren toleranz.

The immune system is regulated by a variety of mechanisms that prevent overwhelming immune responses and a break in tolerance. There have been indications for some time that populations of B cells are also a part of this network and able to exert regulatory functions. Here we summarize current knowledge on this previously unrecognized B cell function and their potential role in protection against autoimmunity.

Contribution of the innate immune system to autoimmune myocarditis: a role for complement.

Myocarditis is a principal cause of heart disease among young adults and is often a precursor of heart failure due to dilated cardiomyopathy. We show here that complement is critical for the induction of experimental autoimmune myocarditis and that it acts through complement receptor type 1 (CR1) and type 2 (CR2). We also found a subset of CD44(hi)CD62L(lo) T cells that expresses CR1 and CR2 and propose that both receptors are involved in the expression of B and T cell activation markers, T cell proliferation and cytokine production. These findings provide a mechanism by which activated complement, a key product of the innate immune response, modulates the induction of an autoimmune disease.

Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells.

Bacterial DNA and synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the immune system in a sequence-dependent manner. Here we investigated the potential of CpG-ODNs to cause proliferation, cytokine production, and regulation of surface molecules in human B-chronic lymphocytic leukemia (CLL) cells. CpG-ODN induced proliferation in both B-CLL cells and normal B cells; however, only B-CLL cells increased proliferative responses when CpG-ODN was added to co-cultures of CD40-ligand transfected mouse fibroblasts (CD40LF) and B cells. Production of interleukin-6 and tumor necrosis factor alpha was detectable at borderline levels, using CpG-ODN as the only stimulus. In contrast, when CpG-ODN was added to co-cultures of B cells and CD40LF, a strong increase in cytokine production occurred in B-CLL cells as well as in normal B cells. The surface molecules CD40, CD58, CD80, CD86, CD54, and MHC class I molecules were up-regulated in B-CLL cells, whereas CD95 expression was not influenced by CpG-ODN stimulation. The same pattern of surface molecule regulation was observed in normal B cells, but up-regulation of CD40 was significantly stronger in B-CLL cells. Costimulation with CpG-ODN and CD40LF resulted in further up-regulation of CD58, CD80, CD86, and MHC class I molecules. In contrast, CD95 expression induced by CD40-ligation was inhibited by CpG-ODN. CpG-ODN activated B-CLL cells acquired a strong stimulatory capacity toward T cells in allogeneic mixed lymphocyte reaction. This effect was completely inhibited by a combination of anti-CD80 and anti-CD86 monoclonal antibody. Taken together, these findings suggest the possible use of CpG-ODN for immunotherapeutic strategies in patients with B-CLL.

IFN-alpha stimulates proliferation and cytokine secretion of CD40-stimulated B cell chronic lymphocytic leukemia cells in vitro.

Interferon (IFN)-alpha has a therapeutic effect in several B cell malignancies, including low-grade non-Hodgkin's lymphoma (NHL), multiple myeloma, and hairy cell leukemia, whereas its efficacy in the treatment of B cell chronic lymphocytic leukemia (B-CLL) is rather limited. In the present study, we investigated the effect of IFN-alpha on the biologic functions of B-CLL cells, which were stimulated by cross-linking of the CD40 antigen. In cell samples from 16 B-CLL patients, the addition of IFN-alpha to CD40-stimulated purified B-CLL cells caused a significant increase in [3H]thymidine uptake (p < 0.003). In B-CLL cells maximally activated by CD40 cross-linking and interleukin-2 (IL-2)/IL-10, proliferation was not further enhanced or inhibited by IFN-alpha. In contrast, proliferation of normal tonsillar B cells stimulated by the combination CD40/IL-2/IL-10 was significantly inhibited by IFN-alpha. Because B-CLL activation might be enhanced by induction of the autocrine growth factor tumor necrosis factor-alpha (TNF-alpha), we investigated the effect of IFN-alpha on the secretion of B cell tropic cytokines. In fact, IFN-alpha significantly stimulated the secretion of IL-6 and TNF-alpha in CD40-activated B-CLL cells (p < 0.01). Although exogenous addition of TNF-alpha did not influence activation of CD40-stimulated B-CLL cells, neutralization of TNF-alpha by polyclonal antibodies led to a complete inhibition of CD40-mediated proliferation of B-CLL cells, suggesting that enhanced proliferation and increased cytokine production by CD40-activated B-CLL cells are independent IFN-alpha-mediated events. The studies presented here provide evidence that IFN-alpha mediates costimulatory signals in the context of T cell-mediated B-CLL cell activation.

Autocrine transforming growth factor-beta from chronic lymphocytic leukemia-B cells interferes with proliferative T cell signals.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of noncycling B cells in lymphatic and extralymphatic tissues. In the present study we investigated the possible contribution of TGF-beta, as secreted by CLL-B cells, on this low proliferative state. CLL-B cells were shown to express TGF-beta RNA and to release bioactive TGF-beta into culture supernatants. Antibody neutralization of endogenously secreted TGF-beta increased the proliferation of CLL-B cells as cultured in the presence of IL-2 or IL-4 or in direct contact with activated CD4+ T cells. In these culture systems, addition of exogenous TGF-beta downregulated basal and cytokineinduced proliferation of CLL-B cells. In contrast, neither neutralization of endogeneous TGF-beta, nor addition of exogeneous TGF-beta changed the proliferation of CLL-B cells as cultured in the CD40 system. In order to further explore this differential antiproliferative effect of TGF-beta, cytokine secretion of B cells and of CD4+ T cells as well as surface marker expression of CD4+ T cells were assessed in relation to TGF-beta: There was no negative effect of TGF-beta on autocrine secretion of TNF-alpha or sCD23 by CLL-B cells. Unlike tonsillar B cells, CLL-B cells cultured alone or in the CD40 system did no release significant amounts of IL-6 or IL-8 into supernatants. Secretion of IL-2 or IL-4 by activated CD4+ T cells was higher, when T cells were cocultured with normal tonsillar B cells than with CLL-B cells. The amount of IL-2 or IL-4 released by CD4+ T cells cocultured in direct contact with tonsillar or CLL-B cells was not consistently influenced either by neutralization of endogenous TGF-beta or by addition of TGF-beta. Exogenous TGF-beta did not downregulate expression of CD40L, CD27, CD28, CD54 or mTNF-alpha by T helper cells activated with anti-CD3 or PHA. In conclusion, autocrine secretion of TGF-beta exhibits an antiproliferative effect on CLL-B cells. This effect is most relevant in B cells cultured in direct contact with activated CD4+ T cells suggesting an indirect mode of action.

Direct cellular interaction with activated CD4(+) T cells overcomes hyporesponsiveness of B-cell chronic lymphocytic leukemia in vitro.

The proliferative response of clonal B cells from patients with chronic lymphocytic leukemia (B-CLL) is drastically reduced compared to normal B lymphocytes stimulated via the B cell antigen receptor complex or by CD40 ligation. In the present study we demonstrate that hyporesponsiveness of CLL-B cells can be overcome by stimulatory pathways mediated by activated CD4(+) T cells. In contrast to CD40 ligation, costimulation with activated T cells promotes a proliferative response in CLL-B cells identical to that in normal B cells. Furthermore, coculture with activated T cells improved survival of CLL-B cells in vitro. Differentiation of CLL-B cells into IgM producing cells was promoted, as well. However, the capacity for IgM secretion remained impaired compared to that of normal B cells. For T-cell-mediated B cell activation direct cellular contact with activated T helper cells is absolutely required. Prevention of CD40/CD40L interaction by CD40 antibody caused only partial inhibition of B cell activation, suggesting that additional signals are involved in T-B cell interaction. Whereas interruption of the ligand pairs CD11a/CD54, CD5/CD72, CD27/CD70 had no influence, the addition of CD58 antibody completely inhibited B cell activation by activated T cells. In costimulation with cellular signals the presence of B-cell-tropic cytokines, such as IL-2 and IL-4, was required to optimize B-CLL proliferation, as demonstrated by the use of neutralizing antibodies. We conclude from these results that proliferative hyporesponsiveness by CLL-B cells can be circumvented by antigen-nonspecific signals in addition to CD40 which are mediated by direct contact with activated T helper cells.

Familial retinoblastoma: where and when?

PURPOSE: To determine when patients with a family history of retinoblastoma and previously normal eye exam develop intraocular disease, and where the new retinoblastoma tumors occur. METHODS: A retrospective chart review of retinoblastoma patients. RESULTS: Sixty-two percent of the first eyes (eyes having a previously normal examination) were diagnosed with retinoblastoma by 6 months of age, 90% by 12 months and 100% by 28 months. For the second eye, 27% were identified by 6 months, 64% by 12 months, 91% by 30 months and 100% by 44 months. The younger the age at initial diagnosis of retinoblastoma, the greater the likelihood that tumors will initially be found in the posterior pole. Macular tumors were diagnosed very early (mean 4 months) and once a retinoblastoma focus had appeared in one eye no new tumors developed in the macula of either eye. CONCLUSION: The timing, location, and number of new retinoblastoma tumors follows a predictable pattern.

Hematopoietic growth factors are differentially regulated in monocytes and CD4+ T lymphocytes: influence of IFN-alpha and interleukin-4.

We investigated the influence of interferon-alpha (IFN-alpha) on the synthesis of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) by monocytes and activated T helper cells. IFN-alpha inhibited the production of GM-CSF in unstimulated and lipopolysaccharide (LPS)-activated monocytes to the same extent as was observed in the presence of IL-4. In highly purified CD4+ T cells, which were activated by incubation with immobilized anti-CD3 antibody and anti-CD28, IFN-alpha reduced production of GM-CSF to 47%. In contrast, GM-CSF production in activated T cells was unaffected by exogenously added IL-4. The production of IL-3 by T helper cells was significantly inhibited by IFN-alpha as well. IL-3 production by CD3/CD28-stimulated T helper cells was exclusively enhanced by IL-4. The exogenous addition of IL-4 led to a highly significant increase of IL-3 levels in T cell supernatants to 231% of control cultures (range 137%-605%), whereas other T cell-derived cytokines, such as IFN-gamma and IL-10, failed to influence IL-3 release. The differential role of IL-4 in IL-3 production was confirmed by the addition of anti-IL-4 antibodies to CD3/CD28-stimulated T cells. Neutralizing anti-IL-4 antibody caused a drastic reduction of IL-3 synthesis by activated T cells, whereas GM-CSF production was independent of neutralization of endogenous IL-4. These experiments define IFN-alpha as an inhibitory substance for the production of hematopoietic growth factors by activated immune cells. The influence of IL-4 on cytokine synthesis appears to be cell type specific, thus revealing a differential stimulatory effect on IL-3 production.

Induction of macrophage-inflammatory protein 1alpha (MIP-1alpha) by interferon-alpha.

The biological functions of macrophage-inflammatory protein 1alpha (MIP-1alpha) include the regulation of inflammatory processes and the inhibition of early hematopoiesis. We investigated the regulation of MIP-1alpha in various cell types in response to IFN-alpha and IL-4 by mRNA analysis and examination of MIP-1alpha protein levels in culture supernatants. In peripheral blood mononuclear cells (PBMNCs) and purified monocytes, IFN-alpha induced MIP-1alpha mRNA expression and stimulated MIP-1alpha secretion in LPS-activated monocytes to almost twice the value observed with LPS alone, whereas IL-4 caused a reduction by 80-95%. The stimulatory effect of IFN-alpha on MIP-1alpha production appeared to be dominant over its inhibition by IL-4. This effect was most pronounced after prolonged incubation periods. In fibroblasts obtained from human foreskin, IFN-alpha in combination with IL-1 stimulated MIP-1alpha secretion to more than three times the level obtained with IL-1. Stimulation of MIP-1alpha expression by IFN-alpha may indirectly influence inflammatory processes and the regulation of early hematopoiesis.

Interferon-alpha stimulates production of interleukin-10 in activated CD4+ T cells and monocytes.

In the present study, we investigated the effect of interferon-alpha (IFN-alpha) on the expression of interleukin-10 (IL-10) mRNA and protein synthesis in human monocytes and CD4+ T cells. In mononuclear cells, IFN-alpha induced expression of IL-10 mRNA and further enhanced lipopolysaccharide (LPS)-stimulated IL-10 expression. In purified monocytes, a strong expression of IL-10 mRNA induced by LPS was not further enhanced by IFN-alpha. In highly purified CD4+ T cells, IFN-alpha upregulated IL-10 mRNA upon activation with phytohemagglutinin and phorbol myristate acetate. In purified monocytes, an effect of IFN-alpha on IL-10 protein synthesis was dependent on costimulation with LPS. Maximal stimulation of IL-10 protein by IFN-alpha was seen after prolonged incubation periods of 48 to 96 hours, whereas IFN-gamma reduced IL-10 production in the early incubation period. Similar effects of IFN-alpha were observed in CD4+ T cells activated with CD3 and CD28 monoclonal antibodies. Addition of IFN-alpha caused an increase of IL-10 in culture supernatants of activated T-helper cells of more than 100% after 96 hours of incubation. In contrast, other cytokines, including IFN-gamma and IL-4, had no influence on IL-10 secretion stimulated by CD3 and CD28 in CD4+ T cells. In serum samples of IFN-alpha-treated individuals, we failed to detect an influence of cytokine treatment on IL-10 serum levels, confirming the requirement of additional activating signals for IFN-alpha-mediated effects on IL-10 synthesis. In conclusion, IFN-alpha enhances the late induction of IL-10, which physiologically occurs upon stimulation of monocytes and T cells. Biologically, this effect might enhance the negative-feedback mechanism ascribed to IL-10, which limits inflammatory reactions.