Christopher Eichman

Herpesvirus Entry Mediator Ligand (HVEM-L), a Novel Ligand for HVEM/TR2, Stimulates Proliferation of T Cells and Inhibits HT29 Cell Growth

Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-κB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nm. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-κB-dependent transcription.

Human Calcium-independent Phospholipase A2 Mediates Lymphocyte Proliferation

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requiressn-2-deacylation catalyzed by a class of enzymes known as phospholipases A2 (PLA2, EC3.1.1.4). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA2 as assessed by Western immunoblot. Low levels of 14-kDa PLA2 protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA2 (iPLA2) protein. Calcium-independentsn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA2inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA2 in this proliferative process. Indeed, evaluation of iPLA2 activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA2 levels occurring at the G2/M phase. Addition of the iPLA2 inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF3), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA2 protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA2 plays a key role in the lymphocyte proliferative response.

CKβ‐8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues

We have previously demonstrated that a tartrate‐resistant acid phosphatase (TRAP)‐positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKβ‐8, RANTES, and MIP‐1α elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP‐1β, MCP‐1, MCP‐2, MCP‐3, MCP‐4, HCC‐1, eotaxin‐2, PARC, SLC, ELC) and 3 CXC chemokines (IL‐8, GROα, SDF‐1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG‐63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross‐desensitization studies demonstrate that RANTES and MIP‐1α can partially inhibit the chemotactic response elicited by CKβ‐8. CKβ‐8, the most potent of the active CC chemokines (ECmax 0.1–0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKβ‐8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKβ‐8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKβ‐8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption. J. Cell. Physiol. 183:196–207, 2000.

Cloning and Characterization of a Novel Integrin β3Subunit

We have identified a novel integrin β3 subunit, termed β3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3′-untranslated region of the β3C subunit differs from the previously reported β3A (platelet) and β3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The β3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the β3A gene. HEK 293 cells were stably co-transfected with αV and either β3C (HEKβ3C) or β3A(HEKβ3A). The viability of HEKβ3C cells was lower than that of HEKβ3A cells, and HEKβ3Ccells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both αVβ3A and αVβ3Cisoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKβ3A, HEKβ3C cells failed to adhere to osteopontin, an αVβ3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the β3 integrin in cell adhesion and suggest a potential role for the β3C integrin subunit in modulating cell-matrix interactions.

Inhibition of pro-inflammatory cytokine generation by CTLA4-Ig in the skin and colon of mice adoptively transplanted with CD45RBhi CD4+ T cells correlates with suppression of psoriasis and colitis

Transfer of CD45RBhi CD4 + naïve T cells into severe combined immunodeficient (SCID) mice induces colitis and skin lesions. Recipients treated with cyclosporin A (CsA), CTLA4-Ig, or vehicle were evaluated for weight loss, skin lesions, and cutaneous blood flow. Necropsy, histological, hematological and cytokine analyses were performed at the conclusion of the experiment to confirm the clinical findings. Vehicle-treated mice lost weight and had 100% incidence of skin lesions by 46-days. CsA-treated mice also lost weight, but only 3/8 mice developed mild, clinically evident skin lesions. In contrast, all CTLA4-Ig-treated mice gained weight and did not develop skin lesions. Increase in cutaneous blood flow correlated with the development of skin lesions. Granulocyte numbers, which were high or moderately high in the vehicle- or CsA-treated mice, respectively, remained as low in the CTLA4-Ig-treated group as in untreated mice. IFN-gamma, IL-1beta, and TNF-alpha levels in the gut and skin correlated with the extent of inflammation in both organs. Histology revealed that CTLA4-Ig but not CsA effectively prevented both autoimmune disorders. The ability of CTLA4-Ig to prevent both colitis and skin lesions suggests that CD28-dependent co-stimulation of T cells is critical for generation of pro-inflammatory cytokines and induction of clinical disease in such autoimmune disorders.

Analysis of CD28 Interactions with Its Cognate Counter-Receptors CD80 and CD86

CD28 serves as a co-signalling molecule for T cell activation through binding to its cognate counter-receptors CD80 and CD86, expressed on antigen presenting cells. This report summarizes studies conducted to determine the regions of CD28 which are involved in its interactions with CD80 and CD86, using site directed mutagenesis, CD28 mAb epitope mapping, receptor based adhesion assays and direct binding of Ig-fusion proteins to cell surface receptors. These studies show that, although the same overall region on CD28 may be involved in the interactions with CD80 and CD86, subtle but important differences distinguish recognition by the two molecules. These findings, along with other observations on the differential pattern of expression and tissue distribution of CD80 and CD86, and induction of differential cytokine profiles by the two counter-receptors, support the contention that these molecules play distinct roles in the regulation of immune responses in vivo.