Identification and initial characterization of four novel members of the interleukin-1 family.

Interleukin-1 (IL-1), fibroblast growth factors (FGFs), and their homologues are secreted factors that share a common beta-barrel structure and act on target cells by binding to cell surface receptors with immunoglobulin-like folds in their extracellular domain. While numerous members of the FGF family have been discovered, the IL-1 family has remained small and outnumbered by IL-1 receptor homologues. From expressed sequence tag data base searches, we have now identified four additional IL-1 homologues, IL-1H1, IL-1H2, IL-1H3, and IL-1H4. Like most other IL-1/FGFs, these proteins do not contain a hydrophobic leader sequence. IL-1H4 has a propeptide sequence, while IL-1H1, IL-1H2, and IL-1H3 encode only the mature protein. Circular dichroism spectra and thermal stability analysis suggest that IL-1H1 folds similarly to IL-1ra. The novel homologues are not widely expressed in mammals. IL-1H1 is constitutively expressed only in placenta and the squamous epithelium of the esophagus. However, IL-1H1 could be induced in vitro in keratinocytes by interferon-gamma and tumor necrosis factor-alpha and in vivo via a contact hypersensitivity reaction or herpes simplex virus infection. This suggests that IL-1H1 may be involved in pathogenesis of immune mediated disease processes. The addition of four novel IL-1 homologues suggests that the IL-1 family is significantly larger than previously thought.

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.

Intralesional cytokines in chronic oxazolone-induced contact sensitivity suggest roles for tumor necrosis factor alpha and interleukin-4.

An analysis was conducted of the cytokine profile and inflammatory response in oxazolone sensitized mouse skin. Following exposure to oxazolone, the intralesional production of inflammatory cytokines was demonstrable at the levels of both mRNA and protein. An initial challenge led to a transient increase in tumor necrosis factor-alpha production followed predominately by the T helper (Th)1 cytokine, interferon-gamma. There was a minimal production of interleukin-4, a Th2 cytokine. Continued exposure to oxazolone led to a downregulation of interferon-gamma and an upregulation of interleukin-4 production. A strong relationship was found between interleukin-4 and the inflammatory response, as measured by ear thickness. Similar experiments conducted in mast cell-deficient mice revealed reduced neutrophil influx but only minor changes in cytokine profile. An irritant response induced by chronic exposure of mouse skin to phorbol ester did not reveal any significant interferon-gamma or interleukin-4 response but was characterized by a tumor necrosis factor-alpha response that correlated with the inflammatory response. These observations suggest that the major source of interferon-gamma and interleukin-4 in the oxazolone response may be the infiltrating lymphocytes; whereas the tumor necrosis factor-alpha may result from the local irritation seen with both oxazolone and phorbol ester. At the end of 4 wk of chronic exposure to oxazolone, it was found that serum IgE levels had significantly increased. Histologic analysis of the skin lesion revealed that a mixed infiltrate including eosinophils developed upon repeat exposure to oxazolone. These findings are consistent with an early predominate Th1 response that is reduced and largely replaced with a Th2 response upon chronic T cell activation.

Expression of ST2, an interleukin-1 receptor homologue, is induced by proinflammatory stimuli.

ST2/T1 is an orphan receptor highly homologous to the IL-1 receptor. Using ST2 cDNA, ST2 specific primers, and a polyclonal antibody generated against ST2, the expression of mRNA and protein corresponding to both the soluble and membrane anchored forms of ST2 was studied. ST2 mRNAs were ubiquitously expressed in all the human tissues examined and were induced by cytokines and phorbol esters. Three different species of mRNAs were observed in different human cells and tissues. In contrast, only two species of ST2 mRNAs were observed in murine Balb/c-3T3 cells and no ST2 mRNA was seen in most tissues of normal mice. However, in a murine model where mouse ears are exposed to UVB irradiation leading to inflammation, ST2 mRNA was expressed 48 h post UV exposure. Similarly, in Balb/c-3T3 cells, the expression of soluble ST2 mRNA and protein was induced by pro-inflammatory stimuli such as TNF, IL-1alpha, IL-1beta and PMA in both exponentially growing and quiescent cells. The expression of the membrane ST2, however, remained constant. These data suggest a role for ST2 in inflammation.

Time-related changes in myeloperoxidase activity and leukotriene B4 receptor binding reflect leukocyte influx in cerebral focal stroke.

In previous studies, we have used histological methods to characterize cellular changes, and validated the use of the myeloperoxidase (MPO) activity assay to quantitate increased neutrophil infiltration in ischemic stroke. We also identified increased leukotriene B4 (LTB4) binding sites as a potential marker for neutrophil infiltration into focal ischemic tissue. However, these studies were conducted at only one time-point, 24 h after ischemia. In the present study, we examined the full time-course of MPO activity and LTB4 receptor binding following middle cerebral artery occlusion (MCAO) made permanently (PMCAO) or transiently (160 min followed by reperfusion; TMCAO) in spontaneously hypertensive rats, and compared the results to previously characterized histologic changes in these models. Ischemic and contralateral (control) cortical tissue samples were assayed for MPO (U/g wet wt) and [3H]LTB4 receptor binding (fmol/mg protein). Following PMCAO, MPO activity significantly increased as early as 12 h and continued to increase over the next 5 d (p < 0.05). Following TMCAO, MPO activity was significantly elevated already after only 6 h of reperfusion and also continued to increase over the next 5 d of reperfusion (p < 0.05). LTB4 receptor binding and MPO activity were highly correlated during periods when both measures increased together (i.e., 0.5-5 d; p <0.01). However, by 15 d post-MCAO, LTB4 receptor binding remained elevated after MPO activity levels had returned to normal. This persistent LTB4 binding was associated with the significant gliosis that was characterized previously to persist in these models. The time-course of increased MPO activity and initially increased LTB4 binding post-MCAO correspond very well to our previous histological data that characterized the time-course for leukocyte infiltration under these conditions. Therefore, the increased MPO activity over time was associated with initial neutrophil and later mononuclear cell infiltration into ischemic tissue in these models. In addition, the present studies utilized histochemical analysis to demonstrate peroxidase activity in macrophages within the cerebral infarct following MCAO, thus validating that MPO activity originates from the later infiltrating mononuclear cells in addition to the early infiltrating neutrophils that had been previously characterized in the same manner. TMCAO produces a significantly larger and earlier increase in ischemic cortex MPO activity and a similar later increase in MPO activity compared to PMCAO treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Association between leukotriene B4-induced phospholipase D activation and degranulation of human neutrophils.

We have explored the role of phospholipase D (PLD) activation in leukotriene B4 (LTB4)-induced Ca2+ mobilization and degranulation of human neutrophils. Stimulation of [3H]alkyl-acyl-phosphatidylcholine-labeled neutrophils with LTB4 resulted in a rapid accumulation of [3H]alkyl-phosphatidic acid (PA) as well as a somewhat slower accumulation of [3H]alkyl-diglyceride (DG). In the presence of ethanol, PLD catalyzed a transphosphatidylation reaction in which LTB4 increased [3H]alkyl-phosphatidylethanol formation and simultaneously decreased LTB4-induced PA and DG accumulation. This pattern of lipid metabolism is consistent with the conclusion that LTB4 stimulates PLD activity in human neutrophils. Additional studies in which the extracellular and intracellular concentrations of Ca2+ were varied indicated that maximal LTB4-induced PLD activation was dependent upon Ca2+ and potentiated by inhibitors of protein kinase C. The time-course and concentration-response curves for LTB4-induced PLD activation were different from those for LTB4-induced Ca2+ mobilization, as measured by fura-2 fluorescence. On the other hand, the concentration-response curve for LTB4-induced PLD activation was similar to that for LTB4-induced degranulation. Preincubation of the cells with ethanol inhibited LTB4-induced PA and DG accumulation, as well as degranulation, suggesting that one or both of these metabolites were important for this response. In contrast, ethanol had no effect on LTB4-induced Ca2+ mobilization. Propranolol, an inhibitor of phosphatidate phosphohydrolase, abolished DG accumulation in response to LTB4 but had no effect on degranulation, suggesting that PA is more important than DG as a mediator of degranulation. Taken collectively, these data indicate that LTB4-induced activation of PLD in human neutrophils is mediated by a Ca(2+)-dependent mechanism, but not by protein kinase C. In addition, PLD activation in these cells may induce degranulation, but not Ca2+ mobilization.