Inhibition of Sphingosine-1-phosphate receptors in ischemia reperfusion injured autoimmunity-prone mice.

B6.MRL/lpr mice, an autoimmune strain, have an accelerated injury time course, increased intensity of tissue damage, and increased CD4+ T cell infiltration in the mesenteric ischemia/reperfusion injury model. In this study, the mechanism by which CD4+ T cells were recruited into injured tissue was addressed. Fingolimod (FTY720) was utilized to assess the role of infiltrating CD4+ T cells. FTY720 treatment was more effective in attenuating injury in B6.MRL/lpr mice then in control mice. Reduced CD4+ cell infiltration and tissue injury correlated with decreased neutrophil infiltration and pro-inflammatory cytokine generation. Inhibiting downstream Sphingosine-1-phosphate (S1P) receptor signaling, specifically GαI mediated signaling, did not inhibit injury, suggesting differential utilization of the S1P receptors between control and MRL/lpr strains. Analysis of S1P receptor expression exposed a predominance of S1P2 in the B6.MRL/lpr strain. Reliance on alternate S1P receptors in the autoimmune strain will alter the progress of inflammation and tissue injury.

Antigen binding and idiotype analysis of antibodies obtained after electroporation of heavy and light chain genes encoding phosphocholine-specific antibodies: a model for T15-idiotype dominance.

Antibodies bearing the T15 idiotype dominate the murine primary immune response to phosphocholine (PC). Analysis of antigen binding of antibodies derived from V1:DFL16.1:JH1 (VH1) germline and N region-derived variant heavy (H) chains and kappa 22, kappa 24, and kappa 8 light (L) chains demonstrates that the T15H:kappa 22L (T15) antibody binds PC at least 20-40 times better than other antibodies derived from alternate germline forms of the VH1 H chain and kappa 22, kappa 24, or kappa 8 L chains. To achieve affinities in the same range as the T15 antibody, kappa 24 and kappa 8 L chain-containing antibodies must have H chains derived from variant N region or somatically mutated VH1 genes. Single amino acid differences at the VD junction of the various germline and N region variant VH1 H chains dictate the L chain that can associate with the H chain to produce a PC-specific antibody. Several H:L combinations give rise to T15 or M167 idiotype-positive antibodies that lack specificity for PC, and single amino acid substitutions or insertions at the VH1:D junction result in the loss of T15 or M167 idiotopes. Based on these observations, our data support a molecular model involving both preferential gene rearrangement and antigen-driven B cell selection to explain T15 idiotype dominance in the immune response to PC. In the absence of N region diversification, large numbers of neonatal B cells bearing the T15H:kappa 22L surface immunoglobulin M (sIgM) receptors would be selected and expanded by autologous or environmental PC antigen into the long-lived peripheral B cell pool.

Phorbol ester-induced, cell-cycle-specific, growth inhibition of human B-lymphoma cell lines.

The activation, growth, and differentiation of three B-cell- and one non-B-cell-derived human lymphoma cell lines were examined after treatment with protein kinase C-activating phorbol esters. Treatment with these agents resulted in early activation events similar to those observed in normal B cells. However, in contrast to their growth-promoting effect on normal human B lymphocytes, exposure to these phorbol esters induced profound growth inhibition of the three B-cell-derived lymphoma lines. Maximal inhibition was achieved within 24 hours of culture initiation and could be reversed if the phorbol ester was removed after 12, but not 20, hours in culture. Cell-cycle analysis of phorbol ester-treated lymphoma cells revealed a G1/S block in one line, whereas cells from the other two lines accumulated in G2/M. These data demonstrate that protein kinase C-binding phorbol esters can interrupt the cell cycle in two places in actively dividing human B-lymphoma cells. These findings may prove valuable with regard to potential therapy of human malignant lymphomas.

Analysis of isotype switch variants of a Qa-5 specific hybridoma.

Qa-5 is a Class I MHC Ag expressed on mature T cells and natural killer (NK) cells of mice, and is defined by specific reactivity with an IgM secreting hybridoma (B16-167). The availability of IgG antibodies with Qa-5 specificity would greatly facilitate in vivo studies of Qa-5 positive NK cells. Using a sequential subline technique in conjunction with ELISA screening, several isotype variants of the B16-167 parent line were selected and cloned. An IgG2a, an IgG2b, and two IgG1 switch variants were selected, and it was demonstrated that one of these cloned lines produced both IgM and IgG1 simultaneously. Binding of the variant antibodies to Qa-5+ cells was not detected by flow cytometry analysis, nor by the ability to deplete NK activity from Qa-5+ large granular lymphocytes (LGL). However, all of the variant lines appeared to use the same VH region genes by Southern blot analysis. Furthermore, IgG2a from the switch variants inhibited binding of the parental IgM anti-Qa-5 when used in excess, and reduction of the parental IgM anti-Qa-5 to monomeric form abolished its binding capacity. These data together indicate that the weak binding by the IgG variants may be due to loss of an innate low affinity/high avidity binding of the parent IgM. Therefore, the isotype switch detection procedure is a useful technique for selection of antibodies with various effector functions, provided the initial antibody is an antibody with sufficient binding affinity.

Anti-IgM-mediated growth inhibition of a human B lymphoma cell line is independent of phosphatidylinositol turnover and protein kinase C activation and involves tyrosine phosphorylation.

The RL cell line is an EBV-negative, surface IgM, IgD-positive B lymphoma line, which is significantly growth arrested in the presence of acrylamide-linked antibodies to the surface IgM receptor. We demonstrate here that activation of protein kinase C (PKC) with PMA abrogates anti-IgM-induced phosphoinositide turnover and Ca2+ mobilization; however, growth inhibition is not affected. In addition, inhibitors of PKC are unable to reverse the anti-IgM-mediated growth inhibition. Two-dimensional gel electrophoresis reveals a different pattern of protein phosphorylation after treatment of RL with PMA or anti-IgM. These data strongly suggest that anti-IgM-induced growth inhibition does not rely on phospholipase C-mediated phosphoinositide turnover, Ca2+ mobilization, or PKC activation. On the other hand, the phosphatase inhibitor orthovanadate results in an augmentation of proteins phosphorylated on tyrosine and the growth inhibition which follows anti-IgM treatment. Furthermore, protein tyrosine kinase inhibitors, genistein and herbimycin A, are able to reverse the anti-IgM-induced inhibition of growth. These data demonstrate that multiple signaling pathways are activated by the interaction of anti-IgM with its ligand, and suggest that tyrosine kinase activation is a critical component of the inhibitory response.