Although TNF is a major proinflammatory cytokine, increasing evidence indicates that TNF also has immunosuppressive feedback effects. We have demonstrated in this study that, in both resting and activated states, mouse peripheral CD4+CD25+ T regulatory cells (Tregs) expressed remarkably higher surface levels of TNFR2 than CD4+CD25− T effector cells (Teffs). In cocultures of Tregs and Teffs, inhibition of proliferation of Teffs by Tregs was initially transiently abrogated by exposure to TNF, but longer exposure to TNF restored suppressive effects. Cytokine production by Teffs remained continually suppressed by Tregs. The profound anergy of Tregs in response to TCR stimulation was overcome by TNF, which expanded the Treg population. Furthermore, in synergy with IL-2, TNF expanded Tregs even more markedly up-regulated expression of CD25 and FoxP3 and phosphorylation of STAT5, and enhanced the suppressive activity of Tregs. Unlike TNF, IL-1β and IL-6 did not up-regulate FoxP3-expressing Tregs. Furthermore, the number of Tregs increased in wild-type mice, but not in TNFR2−/− mice following sublethal cecal ligation and puncture. Depletion of Tregs significantly decreased mortality following cecal ligation and puncture. Thus, the stimulatory effect of TNF on Tregs resembles the reported costimulatory effects of TNF on Teffs, but is even more pronounced because of the higher expression of TNFR2 by Tregs. Moreover, our study suggests that the slower response of Tregs than Teffs to TNF results in delayed immunosuppressive feedback effects.

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-1alpha (SDF-1alpha). Competitive binding studies using 125I-labeled SDF-1alpha 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 x 10(-9) mol/L. These receptors were functional as HUVECs and human aorta endothelial cells (HAECs) migrated toward SDF-1alpha. Although SDF-1alpha-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-1alpha. Furthermore, subcutaneous SDF-1alpha 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-1alpha-induced neovascularization from cross sections of leukocyte-free rat aorta. Taken together, our data indicate that SDF-1alpha 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.

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.

Several lines of evidence indicate the instability of CD4+FoxP3+ regulatory T cells (Tregs). We have therefore investigated means of promoting the stability of Tregs. In this study, we found that the proportion of Tregs in mouse strains deficient in TNFR2 or its ligands was reduced in the thymus and peripheral lymphoid tissues, suggesting a potential role of TNFR2 in promoting the sustained expression of FoxP3. We observed that upon in vitro activation with plate-bound anti-CD3 Ab and soluble anti-CD28 Ab, FoxP3 expression by highly purified mouse Tregs was markedly down-regulated. Importantly, TNF partially abrogated this effect of TCR stimulation and stabilized FoxP3 expression. This effect of TNF was blocked by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. Furthermore, TNF was not able to maintain FoxP3 expression by TNFR2-deficient Tregs. In mouse colitis model induced by transfer of naïve CD4 cells into Rag1−/− mice, the disease could be inhibited by co-transfer of WT Tregs, but not by co-transfer of TNFR2-deficient Tregs. Furthermore, in the lamina propria of the colitis model, the majority of WT Tregs maintained FoxP3 expression. In contrast, increased number of TNFR2-deficient Tregs lost FoxP3 expression. Thus, our data clearly show that TNFR2 is critical for the phenotypic and functional stability of Treg in the inflammatory environment. This effect of TNF should be taken into account when designing future therapy of autoimmunity and GVHD by using TNF inhibitors.

The naphthoquinone pigment, shikonin, isolated from Lithospermum erythrorhizon Sieb. et Zucc.(Boraginaceae) and its derivatives are the active components isolated from the Chinese herbal therapeutic, Zicao. Historically, Zicao root extracts have been used to treat macular eruption, measles, sore-throat, carbuncles and burns. Multiple pharmacological actions have been attributed to shikonin, e.g. antiinflammatory, antigonadotropic and anti-HIV-1 activity. In this review, several therapeutic applications of shikonin will be summarized including its pleiotropic, antiinflammatory and antitumour effects. Widely diverse and sometimes conflicting activities have been attributed to shikonin, e.g. wound healing, enhanced granuloma formation, suppression of local acute inflammatory reactions, inhibition of angiogenesis, inhibition of select chemokine ligands, inhibition of DNA topoisomerase activity, inhibition of platelet activation and antimicrobial activity. Comparison of the various reported mechanisms of action for shikonin lead us to hypothesize that shikonin is an effective inhibitor of protein-protein interaction with multiple targets in both the intracellular and extracellular compartments. This general inhibitory effect can account for the broad spectrum of shikonin biological and pharmacological activities.

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.

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.

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-␣, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.