Amelioration of CR-EAE with lisofylline: effects on mRNA levels of IL-12 and IFN-gamma in the CNS.

CR-EAE is a Th1-mediated inflammatory autoimmune demyelinating disease of the CNS and serves as a model of human multiple sclerosis. Our previous studies have shown the protective effect of orally administered lisofylline in the prevention of active and passively induced acute EAE. In our present studies we have examined the efficacy and mechanism of action of lisofylline on CR-EAE. Lisofylline decreased the number and severity of paralytic attacks in mice with relapsing EAE. The reduction of clinical disease correlated with decreased levels of mRNA levels of IFN-gamma but not of mRNA levels of IL-12. These studies suggest that lisofylline may be an effective therapeutic for established Th1 mediated autoimmune disease and that it acts by blocking IL-12R signaling and not IL-12 production in vivo.

Selective pharmacologic inhibition of murine and human IL-12-dependent Th1 differentiation and IL-12 signaling.

We have previously shown that lisofylline (LSF) inhibits murine Th1-mediated disease in vivo by blocking IL-12-induced differentiation of Th1 cells. The cellular and molecular mechanisms underlying this inhibition were further explored by testing LSF in several IL-12-responsive model systems in vitro. IL-12-dependent Th1 differentiation was abrogated by LSF and yielded effector T cells that were deficient in proinflammatory cytokine secretion, including IFN-gamma, IL-2, and TNF-alpha. The diminished Th1 phenotype resulted from both a lower frequency of IL-12-derived Th1 clones and a reduced capacity of individual clones to secrete IFN-gamma due to lower levels of IFN-gamma mRNA. The arrest in Th1 development resulted from a blockade of IL-12 signaling that preceded the Th0 to Th1 transition. Thus, LSF blocked IL-12-enhanced IFN-gamma production in anti-CD3-stimulated T cells and prevented IL-12-mediated repression of the transcription factor GATA-3. Lisofylline also inhibited IL-12-induced increases in STAT4 tyrosine phosphorylation, but did not block TCR signaling or inhibit acquisition of IL-12 responsiveness. These findings were extended to show that LSF also inhibits IL-12-dependent responses in human T cells. LSF, which has one asymmetric chiral center, was selectively inhibitory for IL-12 signaling compared with its S-enantiomer (1501-S) and the oxidized side chain analog, pentoxifylline. The results suggest that LSF may be useful as a modulator of Th1-mediated disease in humans.

Prevention of experimental allergic encephalomyelitis via inhibition of IL-12 signaling and IL-12-mediated Th1 differentiation: an effect of the novel anti-inflammatory drug lisofylline.

Experimental allergic encephalomyelitis (EAE) is an inflammatory, CD4+ Th1-mediated autoimmune disease, which serves as a model for multiple sclerosis. We examined the effect of a novel anti-inflammatory drug, lisofylline (LSF), on EAE induced either by injection of mouse spinal cord homogenate or following transfer of myelin basic protein-reactive T cells. Orally administered LSF significantly inhibited EAE in both cases, decreasing peak clinical scores by >70% and >80%, respectively. In addition, analysis of representative spinal cord sections from LSF-treated mice showed complete lack of demyelination and lymphocyte infiltration. The reduction in EAE correlated with the inhibition of Th1 differentiation by LSF in vivo, as indicated by a reduction in T cell IFN-gamma production ex vivo after Ag restimulation. The inhibition of Th1 differentiation in vivo is consistent with a block in IL-12 receptor signaling, because LSF blocked IL-12-driven Th1 differentiation and T cell proliferation in vitro, yet had no effect on IL-12 secretion from APCs ex vivo or in vitro.

CD22 regulates thymus-independent responses and the lifespan of B cells.

The B-lymphocyte-restricted glycoprotein CD22 is expressed on mature IgM+IgD+ B cells, and is capable of binding to ligands on T and B cells. CD22 can interact with both the B-cell antigen receptor (BCR) complex and signalling molecules, including the protein tyrosine phosphatase SHP1 (PTP1C, SHP), a putative negative regulator of BCR signalling. Thus CD22 may facilitate interactions with lymphocytes and regulate the threshold of BCR signalling. To define the in vivo function of CD22, we generated CD22-deficient mice. Here we show that CD22 is required for normal antibody responses to thymus-independent antigens and regulates the lifespan of mature B cells.

Functional CD40 ligand expressed by human blood dendritic cells is up-regulated by CD40 ligation.

We examined whether human blood dendritic cells (DCs) express a functional ligand for CD40 (CD40L). Human blood DCs expressed significant amounts of cell surface CD40L identical to that expressed on activated T cells, as detected by mAb to CD40L or a chimeric CD40.Ig fusion protein (CD40.Ig). Stimulation through CD40 up-regulated protein and mRNA CD40L expression in DCs, B cells, and B cell lines. CD40-mediated CD40L expression was inhibited by a protein tyrosine kinase inhibitor, herbimycin, in a dose-dependent manner, suggesting that the induction of CD40L expression via CD40 requires protein tyrosine kinase activity. CD40L surface expression correlated with constitutive or inducible levels of CD40L-specific mRNA, as determined by reverse transcribed PCR analysis (RT-PCR) using CD40L-homologous primers. Furthermore, CD40L on DCs was functional, since CD40L+ DCs, unlike CD40L- DCs, induced B cell IgG and IgA production, and this induction could be inhibited by blocking CD40L-CD40 interactions with mAb to CD40L. Thus, CD40L on DCs and CD40L induced by crosslinking CD40 may regulate B cell activation and maturation. The importance of DC CD40L expression on B cell function is discussed.

Protein kinase C mu (PKC mu) associates with the B cell antigen receptor complex and regulates lymphocyte signaling.

We have identified a Ser/Thr kinase associated with the B cell receptor (BCR) complex as protein kinase C mu (PKC mu). PKC mu activity is up-regulated after cross-linking the BCR and CD19 on B cells, and PKC mu co-precipitates with Syk and phospholipase C-gamma 1/2 (PLC gamma 1/2). In vitro phosphorylation of fusion proteins showed that both Syk and PLC gamma 1 are potential substrates of PKC mu in vivo. Analysis of mutants of the chicken B cell line DT40 deficient in either Syk, Lyn, Btk, or PLC gamma 2 revealed that BCR-induced activation of PKC mu, like activation of PLC gamma 2, requires Syk and is partially regulated by Btk, but is Lyn independent. PKC mu can down-regulate the ability of Syk to phosphorylate PLC gamma 1 in vitro. Thus, PKC mu may function in a negative feedback loop regulating BCR-initiated signaling cascades.

CD45 ligation induces programmed cell death in T and B lymphocytes.

Studies have shown an essential role for the CD45 protein tyrosine phosphatase in regulating Ag receptor-derived signals in lymphocytes. Co-ligating CD45 with the Ag receptor, however, can also inhibit receptor signaling, as manifest in T cells by the lack of IL-2 production and proliferation. We report that CD45 ligation alone induces apoptosis in normal T cells, and that death was greatly potentiated by cross-linking CD3. Normal B cells and T and B cell lines were also induced to die with insoluble CD45 mAb. CD45-induced cell death was blocked by inhibitors of protein tyrosine kinases and protein tyrosine phosphatases, but not by inhibitors of RNA or protein synthesis or by cyclosporin A. Morphologically, CD45-mediated apoptosis resembled death induced via CD95 (Fas), as evidenced by nuclear condensation and membrane blebbing, but did not cause DNA fragmentation into oligonucleosomes. Co-ligating CD45 and CD95 either enhanced or inhibited CD45-induced cell death, depending on the degree of CD45 and CD95 cross-linking. Finally, CD45 cross-linking induced its rapid association with the detergent-insoluble cell fraction, suggesting that it becomes linked to the cytoskeleton during CD45-induced apoptosis. These data show a novel role for CD45 in regulating lymphocyte death as well as proliferation.

Cell-cell interactions regulate dendritic cell-dependent HIV-1 production in CD4+ T lymphocytes.

We investigated the role of blood dendritic cells (DC) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DC promoted transmission from infected to uninfected CD4+ cells, but blood DC themselves were not infectable. DC-mediated transmission was blocked by mAb to CD4 and MHC class II, but strongly increased by mAb to CD40 on DC or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4Ig also blocked infection augmented by crosslinking CD40. We also demonstrated that mAb to CD40 up-regulate the expression of CTLA4 ligands CD80 and B70/B7-2 (CD86) on DC. These data suggest that the dialog between CD40-CD40 ligand (CD40L) and CD28-CD80 counter-receptors on DC and T cells may be linked to HIV infection in vivo.

CD40 and its ligand in the regulation of humoral immunity.

T cells provide help for B cell antibody production by cell-cell contact and by soluble factors. CD40L is the predominant protein induced on activated T cells which constitutes contact-dependent help, and lack of CD40L or blocking CD40-CD40L interactions leads to severely impaired antibody production. In addition to CD40-CD40L, B and T cells express costimulatory, accessory molecules which amplify T and B cell function and allow for reciprocal dialogues during antigen presentation. Interactions between costimulatory counter-receptors can determine lymphocyte activation or nonresponsiveness, and provides a means for regulating self-tolerance.

The role of CD40 and CD80 accessory cell molecules in dendritic cell-dependent HIV-1 infection.

We investigated the role of blood dendritic cells (DCs) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DCs promoted transmission from infected to uninfected CD4+ cells, but DCs themselves were not infectable. DC-mediated transmission was blocked by MAb to CD4 and MHC class II, but strongly increased by MAb to CD40 on DCs or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4 immunoglobulin also blocked infection augmented by cross-linking CD40. These data suggest a linkage between CD40-CD40L and CD28-CD80 counterreceptors on DCs and T cells, and spread of HIV infection in vivo.

Costimulation through CD28 enhances T cell-dependent B cell activation via CD40-CD40L interaction.

Changes in T cell helper function were analyzed when anti-CD3-activated T cells were costimulated with mAbs to the CD28 receptor (anti-CD28). T cell-dependent B cell growth and differentiation were consistently augmented if anti-CD3 stimulated-T cells were simultaneously activated with anti-CD28. Although anti-CD28 enhanced IL-2 and IL-4 production, it did not increase B cell responses solely by augmenting production of soluble lymphokines. Anti-CD28 costimulation induced increases on T cells of CD40 ligand (CD40L), known to promote B cell proliferation and Ig secretion. Because anti-CD28 promoted T cell helper functions and expression of CD40L, we examined the dependence for CD40L during T cell-dependent B cell responses. Although soluble CD40 fusion proteins only partially inhibited T cell-dependent B cell activation, we found a strict requirement for CD40L expression at initiating B cell responses. Both CD40L expression and T cell help were blocked by cyclosporin A after TCR cross-linking, and, unlike T cell proliferation, both remained cyclosporin A sensitive during CD28 costimulation. In addition, anti-CD28 could not compensate for the T cell helper deficiency of hyper IgM syndrome patients who lack functional CD40L. Thus, anti-CD28-induced T cell help is delivered via a CD40L-dependent process. The fact that cross-linking CD40 on B cells promotes expression of the B7/BB-1 ligand for CD28 suggest T and B interactions may have a reciprocal amplification mechanism.

Effects of IL-4 and Fc gamma receptor II engagement on Egr-1 expression during stimulation of B lymphocytes by membrane immunoglobulin crosslinking.

Egr-1 is an immediate early gene that is rapidly upregulated in response to mitogenic signals induced by antigen receptor crosslinking on murine B lymphocytes. It has been shown that levels of Egr-1 expression are closely correlated with B cell proliferation in several models of B cell activation and tolerance. We compared the expression of Egr-1 during B cell stimulation with Fab'2 and IgG anti-immunoglobulin (anti-Ig), since it is known that Fab'2 anti-Ig is mitogenic while IgG anti-Ig is not, owing to a dominant inhibitory effect of crosslinking the B cell Fc gamma RII to membrane Ig. While mitogenic doses of Fab'2 anti-Ig induce large and rapid increases in Egr-1 expression, IgG anti-Ig results in smaller increases in Egr-1 mRNA, comparable to that seen with submitogenic concentrations of Fab'2 anti-Ig. However, the correlation between Egr-1 expression and B cell proliferation breaks down when IL-4 is added as a co-mitogen to induce B cell proliferation with IgG anti-Ig or submitogenic concentrations of Fab'2 anti-Ig. No corresponding increases in Egr-1 mRNA levels are observed when IL-4 is added. Therefore, IL-4 overcomes Fc receptor-mediated inhibition of B cell proliferation without affecting inhibition of Egr-1 mRNA induction, as demonstrated earlier for c-myc mRNA in this system.

Inducible cell contact signals regulate early activation gene expression during B-T lymphocyte collaboration.

B cells get help in the antibody response by presenting processed Ag to Th cells. We asked whether the Ag-presenting B cell must induce Th functions before receiving help, or whether B cell activation is a direct consequence of T cell recognition of Ag on the B cell surface. To obtain a prompt and sensitive indication of the receipt of growth signals, we measured mRNA levels of the immediate early genes, c-myc and egr-1, in T and B cells separated from Ag-specific B-T conjugates of normal, resting murine B cells and a Th line. Although Ag-dependent increases in B cell c-myc expression occur as early as 2 h after conjugation, early c-myc expression in the B cell was also seen when the Th cells were activated with immobilized anti-CD3 in the absence of Ag recognition. Therefore, T cell activation rather than Ag recognition per se appears to be responsible for the early c-myc signal in the B cells. The c-myc response in the B cell depends on induction of a contact-dependent helper function in the T cell, which is inhibitable by cyclosporin A acting on the T cell. Delivery of contact help is not blocked by anti-class II MHC antibody. Contact with activated Th cells induces a different pattern of immediate early gene expression from that induced by cross-linking the B cell Ag receptor.

Window of vulnerability of vaccinia virus-infected cells to natural killer (NK) cell-mediated cytolysis correlates with enhanced NK cell triggering and is concomitant with a decrease in H-2 class I antigen expression.

A time course study was performed in order to determine if vaccinia virus (VV)-infected targets were more susceptible to murine natural killer (NK) cell-mediated lysis during a discrete period of time postinfection. Activated NK cells were used in short-term (e.g. 4 h) assays in order to avoid a further in vitro activation of the NK cells by interferon (IFN) and to test the innate susceptibility of target cells to lysis. The sensitivity of VV-infected L929 cells to lysis by NK cells increased as the infection progressed, reached a peak at approximately 24 h postinfection, and subsequently declined to levels lower than that of uninfected cells. This window of vulnerability was not due to an increase in the number of effector/target cell conjugates, which continually decreased as the VV infection progressed. Triggering of NK cells was measured by the influx of 45Ca2+. Target cells treated with IFN induced less 45Ca2+ uptake, whereas cycloheximide treatment of targets caused a greater influx of 45Ca2+ into the effector cells. When L929 cells were infected with VV for various time intervals and used in the triggering assays, an enhanced triggering of the effectors corresponding to the time of enhanced susceptibility of the target cells to lysis was detected. Quantitative decreases in H-2Kk and Dk class I antigens were observed following VV infection of target cells as measured by FACS analysis using alloantibodies. Qualitative changes in H-2 class I antigens were also observed, as detected by a loss in VV-infected target cell susceptibility to lysis by allospecific cytotoxic T lymphocytes (CTL) at a time when they were highly sensitive to killing by NK cells and VV-specific CTL. These results show that virus-infected targets may become innately more sensitive to lysis by NK cells at discrete time points after infection and that the susceptibility to lysis correlates with enhanced triggering of NK cells and reduced H-2 class I antigen expression.