O. M. Zack Howard

Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Induce Expression of CXCR4 on Human Endothelial Cells : In Vivo Neovascularization Induced by Stromal-Derived Factor-1α

The contribution of chemokines toward angiogenesis is currently a focus of intensive investigation. Certain members of the CXC chemokine family can induce bovine capillary endothelial cell migration in vitro and corneal angiogenesis in vivo, and apparently act via binding to their receptors CXCR1 and CXCR2. We used an RNAse protection assay that permitted the simultaneous detection of mRNA for various CXC chemokine receptors in resting human umbilical vein endothelial cells (HUVECs) and detected low levels of only CXCR4 mRNA. Stimulation of HUVECs with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) up-regulated levels of only CXCR4 mRNA. CXCR4 specifically binds the chemokine stromal-derived factor-1α (SDF-1α). Competitive binding studies using 125I-labeled SDF-1α with Scatchard analysis indicated that VEGF or bFGF induced an average number of approximately 16,600 CXCR4 molecules per endothelial cell, with a Kd = 1.23 × 10−9 mol/L. These receptors were functional as HUVECs and human aorta endothelial cells (HAECs) migrated toward SDF-1α. Although SDF-1α-induced chemotaxis was inhibited by the addition of a neutralizing monoclonal CXCR4 antibody, endothelial chemotaxis toward VEGF was not altered; therefore, the angiogenic effect of VEGF is independent of SDF-1α. Furthermore, subcutaneous SDF-1α injections into mice induced formation of local small blood vessels that was accompanied by leukocytic infiltrates. To test whether these effects were dependent on circulating leukocytes, we successfully obtained SDF-1α-induced neovascularization from cross sections of leukocyte-free rat aorta. Taken together, our data indicate that SDF-1α acts as a potent chemoattractant for endothelial cells of different origins bearing CXCR4 and is a participant in angiogenesis that is regulated at the receptor level by VEGF and bFGF.

Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

Autoantibodies to histidyl–tRNA synthetase (HisRS) or to alanyl–, asparaginyl–, glycyl–, isoleucyl–, or threonyl–tRNA synthetase occur in ∼25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl–tRNA synthetases to induce leukocyte migration. HisRS induced CD4+ and CD8+ lymphocytes, interleukin (IL)-2–activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH2-terminal domain, 1–48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl–tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl–tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl–tRNA and lysyl–tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl–tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.

Glucocorticoid amplifies IL‐2‐dependent expansion of functional FoxP3+CD4+CD25+ T regulatory cells in vivo and enhances their capacity to suppress EAE

IL‐2 is crucial for the production of CD4+CD25+ T regulatory (Treg) cells while important for the generation of effective T cell‐mediated immunity. How to exploit the capacity of IL‐2 to expand Treg cells, while restraining activation of T effector (Teff) cells, is an important and unanswered therapeutic question. Dexamethasone (Dex), a synthetic glucocorticoid steroid, has been reported to suppress IL‐2‐mediated activation of Teff cells and increase the proportion of Treg cells. Thus, we hypothesized that glucocorticoids may be useful as costimulants to amplify IL‐2‐mediated selective expansion of Treg cells. We show in this study that short‐term simultaneous administration of Dex and IL‐2 markedly expanded functional suppressive Foxp3+CD4+CD25+ T cells in murine peripheral lymphoid tissues. In a myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis (EAE) mouse model, we observed that splenic CD4+CD25+ T cells failed to suppress the proliferation of CD4+CD25– T cells. Pretreatment with Dex/IL‐2 remarkably increased the proportion of CD4+FoxP3+ cells and partially restored the function of splenic CD4+CD25+ T cells, and inhibited the development of EAE. Therefore, the combination of glucocorticoid and IL‐2, two currently used therapeutics, may provide a novel approach for the treatment of autoimmune diseases, transplant rejection and graft‐vs.‐host disease.

Cutting Edge: Expression of TNFR2 Defines a Maximally Suppressive Subset of Mouse CD4+CD25+FoxP3+ T Regulatory Cells: Applicability to Tumor-Infiltrating T Regulatory Cells

TNFR2 is predominantly expressed by a subset of human and mouse CD4+CD25+FoxP3+ T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2+ Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30–40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2− Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4+CD25−TNFR2+ cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2+ Tregs was considerably more potent than that of reportedly highly suppressive CD103+ Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2+ Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors.

Differential response of murine CD4+CD25+ and CD4+CD25– T cells to dexamethasone-induced cell death

To evaluate the in vivo effect of immunosuppressive glucocorticoids on CD4+CD25+ T regulatory cells, we injected dexamethasone (Dex) into BALB/c mice. Administrationof Dex enhanced the proportion of CD4+CD25+ cells and the ratio of CD4+CD25+ cells to CD4+CD25– cells in the lymphoid organs, especially in thethymus. This correlates with our in vitro observation that CD4+CD25+ T cells express higher levels of glucocorticoid receptor and Bcl‐2, and are therefore more resistant to Dex‐mediated cell death than CD4+CD25– T cells. Furthermore, IL‐2 selectively protected CD4+CD25+ T cells from Dex‐induced cell death, while IL‐7 and IL‐15 didnot exert preferential protective effects. Dex‐treated CD4+CD25+ T cells expressed higher levels of intracellular CTLA‐4 and surface glucocorticoid‐induced TNF receptor than fresh CD4+CD25+ T cells, but still failed to respond to TCR stimulation and inhibited proliferation of CD4+CD25– T cells. These results suggest that, in addition to suppressing cytokine transcription, Dex treatment is permissive for the survival of functional CD4+CD25+ T regulatory cells, and this property may contribute to the anti‐inflammatory and immunosuppressive efficacy of glucocorticoids. Our data also suggest that selective protection of CD4+CD25+ T cell from apoptosis may constitute a role in immune tolerance for IL‐2.

Prolactin recruits STAT1, STAT3 and STAT5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580.

The present study of prolactin (PRL) receptor-mediated recruitment of signal transducers and activators of transcription (STATs) demonstrates that PRL activates STAT3, in addition to STAT1 and STAT5 as previously reported, and that STAT1, STAT3 and STAT5 are mediators of PRL effects in cells whether of lymphoid, myeloid or mammary epithelial origin. Furthermore, receptor mutants M240 and T280 that do not mediate PRL-induced JAK2 activation and cell proliferation, are also unable to mediate STAT activation, supporting the proposed model of JAK2 as the initial effector protein used by PRL receptors. On the other hand, tyrosine phosphorylation analysis and electrophoretic mobility shift assays showed that receptor mutant G328, which lacks four of the five conserved cytoplasmic tyrosine residues of PRL receptors, retained the ability to activate JAK2 and STAT1, STAT3 and STAT5. These results support the notion that phosphotyrosyl residues other than those of the receptor, i.e., JAK2, are involved in recruiting STAT proteins to the activated PRL receptor complex.

Shikonin, a Component of Chinese Herbal Medicine, Inhibits Chemokine Receptor Function and Suppresses Human Immunodeficiency Virus Type 1

ABSTRACT Shikonin is a major component of zicao (purple gromwell, the dried root of Lithospermum erythrorhizon), a Chinese herbal medicine with various biological activities, including inhibition of human immunodeficiency virus (HIV) type 1 (HIV-1). G protein-coupled chemokine receptors are used by HIV-1 as coreceptors to enter the host cells. In this study, we assessed the effects of shikonin on chemokine receptor function and HIV-1 replication. The results showed that, at nanomolar concentrations, shikonin inhibited monocyte chemotaxis and calcium flux in response to a variety of CC chemokines (CCL2 [monocyte chemoattractant protein 1], CCL3 [macrophage inflammatory protein 1α], and CCL5 [regulated upon activation, normal T-cell expressed and secreted protein]), the CXC chemokine (CXCL12 [stromal cell-derived factor 1α]), and classic chemoattractants (formylmethionyl-leucine-phenylalanine and complement fraction C5a). Shikonin down-regulated surface expression of CCR5, a primary HIV-1 coreceptor, on macrophages to a greater degree than the other receptors (CCR1, CCR2, CXCR4, and the formyl peptide receptor) did. CCR5 mRNA expression was also down-regulated by the compound. Additionally, shikonin inhibited the replication of a multidrug-resistant strain and pediatric clinical isolates of HIV in human peripheral blood mononuclear cells, with 50% inhibitory concentrations (IC50s) ranging from 96 to 366 nM. Shikonin also effectively inhibited the replication of the HIV Ba-L isolate in monocytes/macrophages, with an IC50 of 470 nM. Our results suggest that the anti-HIV and anti-inflammatory activities of shikonin may be related to its interference with chemokine receptor expression and function. Therefore, shikonin, as a naturally occurring, low-molecular-weight pan-chemokine receptor inhibitor, constitutes a basis for the development of novel anti-HIV therapeutic agents.

Chemokines: progress toward identifying molecular targets for therapeutic agents

Leukocyte migration towards injury sites is directed by the interaction of chemokines with their receptors. The stages of migration are closely regulated events that involve chemokine-induced leukocyte adhesion, diapedesis and homing. Current research suggests a pathophysiological role for chemokines in diverse inflammatory states arising from viral, bacterial and parasitic infection, allergic and asthmatic reactions, atherosclerosis and arthritis. A role for chemokines in tumor immunity and angiogenesis has recently been demonstrated. A basis for the rational design of chemokine antagonists is emerging from a knowledge of tertiary structures and mutational analysis of chemokine ligands and receptors. Here, we discuss advances in knowledge about chemokine structure and function, with emphasis on potential therapeutic agents.

Chemotactic Activity of S100A7 (Psoriasin) Is Mediated by the Receptor for Advanced Glycation End Products and Potentiates Inflammation with Highly Homologous but Functionally Distinct S100A15

Human S100A7 (psoriasin) is overexpressed in inflammatory diseases. The recently discovered, co-evolved hS100A15 is almost identical in sequence and up-regulated with hS100A7 during cutaneous inflammation. The functional role of these closely related proteins for inflammation remains undefined. By generating specific Abs, we demonstrate that hS100A7 and hS100A15 proteins are differentially expressed by specific cell types in the skin. Although highly homologous, both proteins are chemoattractants with distinct chemotactic activity for leukocyte subsets. We define RAGE (receptor for advanced glycation end products) as the hS100A7 receptor, whereas hS100A15 functions through a Gi protein-coupled receptor. hS100A7-RAGE binding, signaling, and chemotaxis are zinc-dependent in vitro, reflecting the previously reported zinc-mediated changes in the hS100A7 dimer structure. When combined, hS100A7 and hS100A15 potentiate inflammation in vivo. Thus, proinflammatory synergism in disease may be driven by the diverse biology of these almost identical proteins that have just recently evolved. The identified S100A7 interaction with RAGE may provide a novel therapeutic target for inflammation.

Co‐expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood

Previously, we found that co‐expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3+ cells present in CD25high, CD25low and even CD25– subsets of CD4+ cells expressed high levels of TNFR2. Consequently, TNFR2‐expressing CD4+CD25+ Treg included all of the FOXP3+ cells present in the CD4+CD25high subset as well as a substantial proportion of the FOXP3+ cells present in the CD4+CD25low subset. Flow cytometric analysis of PB identified five‐fold more Treg, determined by FOXP3 expression, in the CD4+CD25+TNFR2+ subset than in the CD4+CD25high subset. In addition, similar levels of FOXP3+ cells were identified in both the CD4+CD25+TNFR2+ and CD4+CD25+CD127low/− subsets. Furthermore, the CD4+CD25+TNFR2+ subset expressed high levels of CTLA‐4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4+CD25+TNFR2+ cells were anergic and markedly inhibited the proliferation and cytokine production of co‐cultured T‐responder cells. In contrast, CD4+CD25+TNFR2– and CD4+CD25–TNFR2+ T cells did not show inhibitory activity. As some non‐Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.