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The cytokine-induced C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is an important regulator of leukocyte recruitment to sites of inflammatory challenge. Here, it is demonstrated that the widely distributed contact hapten NiCl(2), like tumor necrosis factor alpha (TNFalpha), induces monocyte-chemoattractant activity in primary human endothelial cells via induction of MCP-1. NiCl(2) rapidly activated mitogen-activated protein (MAP) kinase p38, and inhibition of p38 partially blocked NiCl(2)-induced MCP-1 messenger RNA and protein expression. Both NiCl(2)- and TNFalpha-induced MCP-1 synthesis was sensitive to D609, an inhibitor of phosphatidylcholine-dependent phospholipase C (PC-PLC). NiCl(2)-induced MCP-1 synthesis required activation of NF-kappaB since mutation of NF-kappaB-binding sites in the promoter resulted in complete loss of inducible promoter activity. Consistent with that finding, stimulation with NiCl(2) or TNFalpha activated IkappaB kinase-beta (IKKbeta), and transient transfection of dominant-negative IKKbeta strongly inhibited NiCl(2)- and TNFalpha-induced MCP-1 expression. However, D609 and the specific p38 inhibitor SB202190 did not affect NiCl(2)- and TNFalpha-induced IKKbeta activation, NF-kappaB DNA-binding activity, or transcriptional activity of a Gal4p65 fusion protein. This indicates that p38- and PC-PLC-dependent pathways directly regulate the transcriptional activity of NF-kappaB factors in the transcriptional complex. Consistent with that, inhibition of p38 blocked enhanced transcriptional activity induced by the transcriptional coactivator p300. Thus, it was concluded that at least 3 independent pathways regulate MCP-1 expression in endothelial cells. Its induction requires activation of the IKKbeta/IkappaBalpha/NF-kappaB signaling pathway, resulting in nuclear accumulation of p65 and subsequent recruitment of cofactors. Proper assembly and activity of this transcriptional complex is further modulated by the p38 MAP kinase cascade and a PC-PLC-dependent pathway.

MAPKs are crucially involved in the regulation of growth and differentiation of a variety of cells. To elucidate the role of MAPKs in keratinocyte differentiation, activation of ERK, JNK, and p38 in response to stimulation with extracellular calcium was analyzed. We provide evidence that calcium-induced differentiation of keratinocytes is associated with rapid and transient activation of the Raf/MEK/ERK pathway. Stimulation of keratinocytes with extracellular calcium resulted in activation of Raf isozymes and their downstream effector ERK within 10-15 min, but did not increase JNK or p38 activity. Calcium-induced ERK activation differed in kinetics from mitogenic ERK activation by epidermal growth factor and could be modulated by alterations of intracellular calcium levels. Interestingly, calcium stimulation led to down-regulation of Ras activity at the same time that ERK activation was initiated. Expression of a dominant-negative mutant of Ras also did not significantly impair calcium-induced ERK activation, indicating that calcium-mediated ERK activation does not require active Ras. Despite the transient nature of ERK activation, calcium-induced expression of the cyclin-dependent kinase inhibitor p21/Cip1 and the differentiation marker involucrin was sensitive to MEK inhibition, which suggests a role for the Raf/MEK/ERK pathway in early stages of keratinocyte differentiation.

Background & Aims: Appropriate management of primary gastric lymphoma is controversial. This prospective, multicenter study aimed to evaluate the accuracy of endoscopic biopsy diagnosis and clinical staging procedures and assess a treatment strategy based on Helicobacter pylori status and tumor stage and grade., Methods: Of 266 patients with primary gastric B-cell lymphoma, 236 with stages EI (n = 151) or EII (n = 85) were included in an intention-to-treat analysis. Patients with H. pylori-positive stage EI low-grade lymphoma underwent eradication therapy. Nonresponders and patients with stage EII low-grade lymphoma underwent gastric surgery. Depending on the residual tumor status and predefined risk factors, patients received either radiotherapy or no further treatment. Patients with high-grade lymphoma underwent surgery and chemotherapy at stages EI/EII, complemented by radiation in case of incomplete resection., Results: Endoscopic-bioptic typing and grading and clinical staging were accurate to 73% and 70%, respectively, based on the histopathology of resected specimens. The overall 2-year survival rates for low-grade lymphoma did not differ in the risk-adjusted treatment groups, ranging from 89% to 96%. In high-grade lymphoma, patients with complete resection or microscopic tumor residuals had significantly better survival rates (88% for EI and 83% for EII) than those with macroscopic tumor residues (53%; P < 0.001)., Conclusions: There is a considerable need for improvement in clinical diagnostic and staging procedures, especially with a view toward nonsurgical treatment. With the exception of eradication therapy in H. pylori-positive low-grade lymphoma of stage EI and the subgroup of locally advanced high-grade lymphoma, resection remains the treatment of choice. However, because there is an increasing trend toward stomach-conserving therapy, a randomized trial comparing cure of disease and quality of life with surgical and conservative treatment is needed.

Bacterial endotoxin (lipopolysaccharide, or LPS) has potent proinflammatory properties by acting on many cell types, including endothelial cells. Secretion of the CXC-chemokine interleukin-8 (IL-8) by LPS-activated endothelial cells contributes substantially to the inflammatory response. Using human umbilical vein endothelial cells (HUVECs), we analyzed the role of small GTP-binding Rho proteins and p38 mitogen-activated protein kinase (MAPK) for LPS-dependent IL-8 expression in endothelial cells. Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced tyrosine phosphorylation, nuclear factor (NF)-kappaB-dependent gene expression, IL-8 messenger RNA, and IL-8 protein accumulation but showed no effect on LPS-dependent p38 MAPK activation. Inhibition of p38 MAPK by SB 202190 also blocked LPS-induced NF-kappaB activation and IL-8 synthesis. Furthermore, selective activation of the p38 MAPK pathway by transient expression of a constitutively active form of MAPK kinase (MKK)6, the upstream activator of p38, was as effective as LPS with respect to IL-8 expression in HUVECs. In summary, our data suggest that LPS-induced NF-kappaB activation and IL-8 synthesis in HUVECs are regulated by both a Rho-dependent signaling pathway and the MKK6/p38 kinase cascade.

A 29-year-old female patient with an autoimmune subepidermal blistering disease had linear deposits of both IgA and IgG at the basement membrane zone. Clinically, the patient presented with tense blisters on the face, trunk, extremities and oral mucosa. Histologically, we found a subepidermal blister formation and a predominantly neutrophilic infiltrate. Direct immunofluorescence showed linear deposits of IgA along the basement membrane zone, as well as linear deposits of IgG and C3 as typically found in bullous pemphigoid. Indirect immunofluorescence demonstrated circulating IgA and IgG autoantibodies. This case extends previous reports on a subgroup of patients with subepidermal blistering diseases characterized by the presence of both IgA and IgG anti-basement membrane antibodies. These patients reveal clinical, histological and immunopathological features of linear IgA disease and bullous pemphigoid.

Human malignant melanoma (MM) is a highly aggressive tumour which is particularly prone to specific local immune responses. To determine the microanatomical location and the species of chemokines possibly involved in the intricate control of cell migration and positioning of immune effector cells in primary and metastatic MM lesions, the expression of those chemokines with lymphocyte and/or macrophage chemoattractant properties was analysed by in situ hybridization. GROalpha (growth-related oncogene) and IL-8 (interleukin 8) were expressed at low levels by single melanoma cells, adjacent keratinocytes, and infiltrating leukocytes. In contrast, the lymphocyte-specific chemokine Mig (monokine induced by interferon-gamma) was strongly expressed by mononuclear cells (mainly macrophages) infiltrating the tumour margin in primary MM lesions, whereas expression was less intense in MM metastasis. IP-10 (interferon-gamma inducible protein 10) was expressed in the same loci at lower intensity. Marked infiltration of T cells was exclusively detected in those areas which exhibited strong Mig expression, whereas areas in the vicinity of tumour cells devoid of Mig expression were not infiltrated. In contrast to Mig, expression of MCP-1 (macrophage chemotactic protein-1) was weaker and mainly detected in lesional basal keratinocytes, occasionally at sites of macrophage infiltration, as well as in single melanoma cells. MIP-1alpha (macrophage inflammatory protein 1alpha) showed similar, albeit weaker expression compared with MCP-1. Other chemokines relevant for the recruitment of monocytes and lymphocytes, such as RANTES (regulated on activation, normal T cells expressed and secreted) and MIP-1beta, were barely detectable. In summary, the chemokine expression profiles support the notion that particularly in heavily infiltrated primary MM lesions, Mig and to a lesser extent IP-10 are important mediators of an IFN-gamma-dependent pathway. Due to their lymphoattractant properties and the known inhibitory effects on the tumour vasculature, both chemokines may be critical for the control of local melanoma tumour growth., (Copyright 1999 John Wiley & Sons, Ltd.)

The glycosaminoglycan hyaluronan, which supports tumor cell migration and metastasis, interferes with fibrin polymerization and leads to increased fiber size and porosity of fibrin clots. Here we have studied the proportionate effect of fibrin polymerization on hyaluronan-mediated migration of glioblastoma cells. The structural and physical properties of hyaluronan-containing fibrin gels were analyzed by turbidity measurement, laser scanning microscopy, compaction assay, and calculation of pore size by liquid permeation. When fibrin polymerized in the presence of hyaluronan or dextran, the resulting gels strongly stimulated cell migration, and migration significantly correlated with fiber mass-to-length ratios and pore diameters. In contrast, cell migration was not induced by addition of hyaluronan to supernatants of already polymerized gels. Hyaluronan-mediated migration was inhibited in fibrin gels by antibodies to alphav- and beta1integrins and the disintegrin echistatin, but not by antibodies to the hyaluronan receptor CD44 (up to 50 microg/ml). As a control, we show that anti-CD44 (10 microg/ml) inhibited cell migration on a pure hyaluronan matrix using a two-dimensional Boyden chamber system. In contrast to three-dimensional migration, the migration of cells on the surfaces of variably structured fibrin gels was not significantly different, indicating that increased gel permeability (porosity) may account for hyaluronan-mediated migration. We conclude that, in complex three-dimensional substrates, the predominant effect of hyaluronan on cell migration might be indirect and requires modulation of fibrin polymerization.

Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C subfamily of chemokines, is important for the local recruitment of leukocytes to sites of inflammatory challenge. Here, we investigated endothelial signaling pathways involving members of the mitogen-activated protein (MAP) kinase superfamily and studied their role for MCP-1 expression in endothelium. We show that tumor necrosis factor-alpha (TNF-alpha), a potent inflammatory activator of endothelium, leads to activation of MAP kinases ERK, p38, and JNK in human umbilical vein endothelial cells (HUVEC). Contribution of MAP kinase pathways to TNF-alpha-induced synthesis of endothelial MCP-1 was then studied by pharmacologic inhibition and transient expression of dominant negative or constitutively active kinase mutants using flow cytometry, Northern blot, and luciferase reporter gene assays. Inhibition of Raf/MEK/ERK or SEK/JNK pathways had no significant effect on MCP-1 levels, whereas blocking the MKK6/p38 pathway by p38 inhibitors SB203580 or SB202190 or by a dominant negative mutant of MKK6, the upstream activator of p38, strongly inhibited TNF-alpha-induced expression of MCP-1. Consistent with that finding, expression of wild-type or constitutively active MKK6 significantly enhanced the effect of limiting TNF-alpha concentrations on MCP-1 synthesis. These data suggest a crucial role for the MKK6/p38 stress kinase cascade in TNF-alpha-mediated endothelial MCP-1 expression.

Dense accumulation of mononuclear cells (lymphocytes >> macrophages) in the dermal-epidermal interface and a T cell-mediated cytotoxic reaction against basal keratinocytes are hallmarks of lichen planus lesions. In this study, we focused on the chemotactic signals responsible for the selective recruitment of these cells. Using in situ hybridization and immunohistochemistry, the expression and localization of the lymphocyte-and/or monocyte/macrophage-attractant CC chemokines macrophage chemoattractant protein-1 (MCP-1), regulated on activation, normal T cell expressed, and secreted (RANTES), macrophage inflammatory protein-1alpha and -1alpha (MIP-1alpha/beta), I-309 and the CXC chemokines monokine induced by interferon-gamma (MIG), interferon-gamma-inducible protein-10 (IP-10), interleukin-8 (IL-8), epithelial-derived neutrophil attractant-78, and growth-related oncogene-alpha were investigated. Strong mRNA expression of MIG, IP-10, and MCP-1 and moderate mRNA expression of RANTES and MIP-1alpha were detected exclusively within foci characterized by strong infiltration with CD3+ lymphocytes (CD4+ cells > CD8+ cells) and CD68+ macrophages. All other chemokines investigated were minimally expressed or absent. With more than 11% of total cells strongly expressing MIG transcripts, this selectively lymphotactic chemokine was by far the dominant chemokine and thus may significantly contribute to the inflammatory reaction in lichen planus lesions. According to the mRNA expression profiles, MIG, IP-10, and MCP-1 were expressed by both basal keratinocytes above and mononuclear cells within the inflammatory foci. Our findings indicate that a set of chemokines composed of IP-10, MCP-1, RANTES, MIP-1alpha, and especially MIG contributes to the cytokine network and preferential trafficking of mononuclear cells to the interface region of lichen planus lesions.

Healing of cutaneous wounds requires a complex integrated network of repair mechanisms, including the action of newly recruited leukocytes. Using a skin repair model in adult humans, we investigated the role chemokines play in sequential infiltration of leukocyte subsets during wound healing. At day 1 after injury, the C-X-C chemokines IL-8 and growth-related oncogene alpha are maximally expressed in the superficial wound bed and are spatially and temporally associated with neutrophil infiltration. IL-8 and growth-related oncogene alpha profiles also correlate with keratinocyte migration and subsequently subside after wound closure at day 4. Macrophage infiltration reaches the highest levels at day 2 and is paralleled by monocyte chemoattractant protein-1 mRNA expression in both the basal layer of the proliferative epidermis at the wound margins and mononuclear cells in the wound area. Other monocyte-attracting chemokines such as monocyte chemoattractant protein-3, macrophage inflammatory protein-1alpha and -1beta, RANTES, and 1309 are undetectable. At day 4, perivascular focal lymphocyte accumulation correlates with strong focal expression of the C-X-C chemokines Mig and IP-10. Our results suggest that a dynamic set of chemokines contributes to the spatially and temporally different infiltration of leukocyte subsets and thus integrates the inflammatory and reparative processes during wound repair.

Vascular disease associated with increased blood concentrations of homocysteine has been known for many years. However, the pathobiochemical mechanisms leading to vasculopathy are still unknown. Several attempts have been made to establish in vitro model systems for the evaluation of homocysteine specific effects in cultured cells. It was concluded from these experiments, that hyperhomocysteinemia has to be considered as a risk factor for atherosclerosis exerting its effects mainly by mechanisms involving oxidative damage. Here, we summarize the homocysteine induced cellular effects which may be due to alterations of the redox thiol status. Effects specific for homocysteine are demonstrated working on different levels of cellular processes involving protein folding and regulation of nuclear factor kappaB (NF-kappaB) controlled gene transcription, the latter opening a new perspective for a novel pathway by which homocysteine might enhance chronic inflammation of the endothelium and possibly contributes to the development of atherosclerosis.

Cell response to a wide variety of extracellular signals is mediated by either mitogenic activation of the Raf/MEK/ERK kinase cascade or stress-induced activation of the mitogen-activated protein kinase (MAPK) family members c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) or p38. We have examined communications between these stress- and mitogen-induced signaling pathways. We show here that the stress cascade activator arsenite activates extracellular signal-regulated kinase (ERK) in addition to p38 albeit with different kinetics. Whereas p38 is an early response kinase, ERK activation occurs with delayed time kinetics at 2-4 h. We observed activation of ERK upon arsenite treatment in many different cell lines. ERK activation is strongly enhanced by overexpression of p38 and mitogen-activated protein kinase kinase 6 (MKK6) but is blocked by dominant negative kinase versions of p38 and MKK6 or the specific p38 inhibitor SB203580. Arsenite-induced ERK activation is mediated by Ras, Raf, and MEK but appears to be independent of de novo protein synthesis. These data provide the first evidence for a p38 dependent activation of the mitogenic kinase cascade in stress-stimulated cells.

A prominent feature within the histopathological changes of psoriatic lesions is the particular spatial distribution of neutrophils, macrophages, and T-cell which are considered to participate in the pathogenesis of psoriasis. In this study, an attempt has been made to examine the microanatomical localization and magnitude of expression of the T-cell-attractant and -stimulating C-X-C and C-C chemokines Mig, interferon-inducible protein-10 (IP-10), macrophage inflammatory protein-1 alpha and 1 beta (MIP-a alpha and 1 beta), and regulated on activation, normal T-cell expressed and secreted (RANTES). Employing in situ hybridization, Mig message was strongly and selectively expressed in the upper lesional dermis with pronounced clustering in the tips of the papillae, whereas expression in normal or uninvolved skin was quiescent. In contrast, message for all the other chemokines investigated was much weaker or lacking. Expression of Mig transcripts in cell clusters of the papillae was paralleled by Mig immunoreactivity on endothelial and mononuclear cells. The expression profile, with high levels of Migs virtually limited to those lesional papillae with a pronounced infiltration of mononuclear leukocytes, strongly suggests that Mig is produced by a local population of highly activated macrophages and dermal microvascular endothelial cells. Considering the T-cell-attracting and -stimulating capacity of Mig and the importance of T-cells in the pathogenesis of psoriasis, this study indicates that this novel C-X-C chemokine plays an important role as a mediator of T-cell recruitment and activation in the papillae and thus contributes significantly to the cytokine network of inflammation in psoriasis.

Chemokines secreted by endothelium have been demonstrated to promote leucocyte recruitment to sites of inflammation. In the present study we investigated the effect of the T lymphocyte-secreted cytokine interleukin (IL)-13 on endothelial expression of chemokines. Employing in situ hybridization and enzyme-linked immunosorbent assay (ELISA) techniques we demonstrate that IL-13, which shares many of its activities with IL-4, selectively induces expression of the C-C chemokine monocyte chemoattractant protein (MCP)-1 in human umbilical vein endothelial cells (HUVEC). However, it fails to up-regulate other C-C and C-X-C chemokines potentially inducible in endothelium such as RANTES (regulated on activation, normal T expressed and secreted), gro-alpha, or IL-8. IL-13 dose-dependently induces monocyte chemotactic activity by HUVEC which can be efficiently blocked by neutralizing antisera against MCP-1. In contrast to the synergistic effect of IL-13 and tumour necrosis factor-alpha (TNF-alpha) on endothelial vascular cell adhesion molecule-1 (VCAM-1) surface expression, TNF-alpha-induced secretion of MCP-1 is not augmented by IL-13. Studying the signalling pathway activated by IL-13 it is demonstrated that a neutralizing monoclonal antibody (mAb) to the 140,000 MW component of the IL-4 receptor (IL-4R alpha) inhibits the effect of IL-13. Immunoprecipitation studies reveal that endothelial IL-4R alpha is rapidly tyrosine phosphorylated upon treatment with IL-13 and IL-4. We furthermore show that both cytokines activate the signal transducer and activator of transcription (Stat) protein-6 in endothelial cells. Our data suggest that IL-13 partly utilizes components of the IL-4 receptor signalling pathway for induction of endothelial MCP-1 expression to facilitate recruitment of blood leucocytes.

Myeloid-related protein (MRP) 8 and MRP14, two members of the S100 family expressed in myelomonocytic cells, have been ascribed some extracellular functions, e.g. antimicrobial, cytostatic, and chemotactic activities. Since S100 proteins lack structural requirements for secretion via the classical endoplasmic reticulum/Golgi route, the process of secretion is unclear. We now demonstrate the specific, energy-dependent release of MRP8 and MRP14 by human monocytes after activation of protein kinase C. This secretory process is not blocked by inhibitors of vesicular traffic through the endoplasmic reticulum and Golgi, and comparative studies on tumor necrosis factor-alpha and interleukin-1beta indicate that MRP8 and MRP14 follow neither the classical nor the interleukin-1-like alternative route of secretion. Inhibition by microtubule-depolymerizing agents revealed that MRP8/MRP14 secretion requires an intact tubulin network. Accordingly, upon initiation of MRP8/MRP14 secretion, immunofluorescence microscopy showed a co-localization of both proteins with tubulin filaments. Release of MRP8 and MRP14 is associated with down-regulation of their de novo synthesis, suggesting that extracellular signaling via MRP8/MRP14 is restricted to distinct differentiation stages of monocytes. Our data provide evidence that the S100 proteins MRP8 and MRP14 are secreted after activation of protein kinase C via a novel pathway requiring an intact microtubule network.

Activation of endothelium is a critical event during the initiation of inflammatory processes and is associated with the induction of cell adhesion molecules and cytokines. The latter include chemotactically active cytokines (chemokines) that promote leukocyte diapedesis from the circulation to sites of evolving inflammation. In this study we evaluated the chemokine repertoire of human endothelial cells derived from the skin (HDMECs) and regulation of these chemokines by cytokines. HDMECs and an immortalized human dermal microvascular endothelial cell line, HMEC-1, were investigated for the expression of C-X-C and C-C chemokines at mRNA and protein levels. Upon stimulation with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), both HDMECs and HMEC-1 expressed high levels of IL-8, GRO, and monocyte chemoattractant protein-1 (MCP-1). RANTES was only weakly induced; however, concomitant treatment with TNF-alpha and interferon-gamma (IFN-gamma) led to upregulation of RANTES, indicating a synergy between these two cytokines. The C-X-C chemokine IFN-inducible protein-10 was upregulated by IFN-gamma but not by other cytokines studied. Macrophage inflammatory protein-1alpha and beta, 1-309, and ENA-78 could not be induced. The chemokine repertoires of HDMECs and HMEC-1 were compared to those of human umbilical vein endothelium and found to be rather similar with the important exception that IFN-gamma and IL-4 up-regulated MCP-1 only in macrovascular endothelium. Our data indicate that HDMECs contribute to the dermal cytokine network by selective production of MCP-1, IL-8, GRO, RANTES, and IP-10, which may critically influence the site-specific recruitment of leukocyte subsets.

Dense focal accumulation of neutrophils in the upper epidermis is a hallmark of psoriasis. Because the signals for neutrophil diapedesis and migration in vivo are not fully understood, psoriatic lesions with pronounced migration of neutrophils may serve as an important model for studying neutrophil chemotaxis. In this study, we present evidence for differential expression of the neutrophil chemotactic cytokines growth-related oncogene alpha, interleukin-8, and ENA-78 (epithelial cell derived and neutrophil-activating properties, 78 amino acids) in psoriatic lesions. In situ hybridization and immunohistochemistry of serial sections were employed to identify and microanatomically localize the cells producing these chemokines. High levels of focal interleukin-8 message were found to be expressed in the upper epidermis by keratinocytes and, most importantly, neutrophils themselves. Growth-related oncogene alpha transcripts were detected in clusters of keratinocytes of the upper epidermis at the same sites where interleukin-8 mRNA was abundant. In contrast to interleukin-8, growth-related oncogene alpha was also detected in the papillary dermis produced by vessel-associated cells. Sites of interleukin-8 and growth-related oncogene alpha mRNA expression were associated with infiltration of neutrophils. Interestingly, mRNA expression of the highly homologous chemokine ENA-78 was quiescent. In conclusion, our data indicate that growth-related oncogene alpha is an important chemoattractant for neutrophil diapedesis in vivo, whereas further migration of neutrophils and formation of micropustules appears to be influenced by the cooperative action of both growth-related oncogene alpha and interleukin-8.

E-selectin (CD62E, formerly termed ELAM-1) is a cytokine-inducible adhesion molecule which mediates the binding of neutrophils, monocytes, and skin homing T-cells. The murine homologue of E-selectin has been cloned. A monoclonal antibody (21KC10) was used here to study immunohistochemically the expression and regulation of murine E-selectin in vitro and in vivo. As described for the human system, there was no staining of normal endothelium in skin and other tissues. LPS and tumour necrosis factor-alpha (TNF-alpha), but not interleukin-4 (IL-4) or interferon-gamma (IFN-gamma), induced a transient expression of E-selectin, both when injected in vivo and when added to endothelial cell lines in vitro. To analyse temporal expression of E-selectin under pathophysiological conditions in vivo, we chose two murine models of inflammation: allergic (ACD) and irritant contact dermatitis (ICD). Expression of E-selectin was found to be induced on vascular endothelium of post-capillary venules in both ACD and ICD. In ICD, maximal staining of endothelial cells occurred earlier than in ACD. Expression of E-selectin during ICD and ACD was then compared between strains of mice which differ with regard to the intensity of their inflammatory reaction. BALB/c mice, which in contrast to C57BI/6 mice show a denser infiltrate and prolonged influx of granulocyte and monocytes, revealed a more pronounced and more prolonged expression of E-selectin than C57BI/6 mice. This held true for both ACD and ICD, and in each case, peak expression of E-selectin was associated with the highest density of the leukocytic infiltrate. This study thus reveals regulatory mechanisms involved in the expression of murine E-selectin in vivo and in vitro. It also demonstrates a correlation between endothelial expression of E-selectin and the genetically determined intensity of the inflammatory response.

Recent evidence indicates that CD44, a multifunctional adhesion receptor involved in cell-cell as well as in cell-matrix interactions, plays an important role in local progression and metastasis of malignant tumors. We have studied a set of human melanoma cell lines differing in their metastatic potential in nude mice as well as in normal melanocytes for changes in CD44 expression and function. All melanocytes and melanoma cell lines tested highly expressed the CD44 standard form (CD44s, 85 kDa) but variants at low levels only. With respect to one of the CD44-associated functions primarily involved in tumor progression we found that two highly metastatic tumor cell lines, MV3 and BLM, showed fivefold higher migration rates towards hyaluronate than melanomas with low metastatic potential and normal melanocytes. Moreover, the highly metastatic cell lines expressed four- to sixfold higher levels of the CD44 epitope involved in hyaluronic acid-binding (monoclonal antibody Hermes-1) than less aggressive melanomas and melanocytes. Hermes-1 efficiently blocked haptotaxis to hyaluronate, supporting the functional relevance of this epitope. In contrast, expression levels of other CD44s epitopes recognized by seven different anti-CD44 monoclonal antibodies were unchanged, suggesting that the migratory behaviour of the cells depends on the formation of the hyaluronate-binding Hermes-1 epitope rather than on the overall CD44s surface expression, which was virtually identical in all melanoma and melanocyte cell lines tested. Differences in the accessibility of the hyaluronate-binding epitope defined by Hermes-1 correlated with the phosphorylation state of CD44s, probably reflecting different activation states of the receptor. Furthermore, immunoprecipitation and pulse/chase studies revealed a three- to fivefold increase in CD44 synthesis in the highly aggressive melanoma cells as compared to the other cell lines and the melanocytes, indicating a reduction of CD44 half-life and up-regulation of turnover. Moreover, highly aggressive melanoma cell lines were found to shed significant amounts of CD44 from the cell surface and to secrete its ligand hyaluronic acid, which may refer to an "autocrine' mechanism mediating melanoma cell motility.

VCAM-1 (vascular cell adhesion molecule-1) is a cytokine-inducible adhesion molecule which is known to mediate adhesion of mononuclear cells to endothelial cells in vitro via binding to the integrin VLA-4 (very late antigen-4). To further elucidate the role and regulation of VCAM-1 in vitro, we compared in vitro and in vivo expression of VCAM-1 in response to cytokines and investigated immunohistochemically the expression of VCAM-1 in three murine models of experimental inflammation. These models differed with regard to the pathogenetic mechanism, and the subsesquent infiltrate: allergic contact dermatitis (ACD) to DNFB as a T cell-controlled, DTH type of inflammation, cutaneous infection with Leishmania major as a chronic granulomatous inflammation and the cauterized cornea as a model for acute inflammation. VCAM-1 was found to be markedly enhanced on vascular endothelia in all types of inflammation and after subcutaneous administration of LPS and TNF-alpha. Administration of IL-4, however, failed to induce VCAM-1 both in vivo and in vitro. The increased VCAM-1 expression in the inflammatory models correlated with the appearance of infiltrating monocytes/macrophages. A concomitant influx of CD4-positive/CD8-positive lymphocytes was only observed in ACD and Leishmaniasis.

Nickel chloride (NiCl2) and cobalt chloride (CoCl2), two haptens frequently leading to contact hypersensitivity in industrialized countries, induce gene transcription of adhesion molecules ICAM-1, VCAM-1, and E-selectin in endothelial cells. In search of transcriptional mechanisms underlying their gene-inductive effects, we studied the capacity of both haptens to activate nuclear factor (NF)-kappa B, a transcription factor involved in inducible expression of adhesion molecules. Using electrophoretic mobility shift assays, a strong increase of NF-kappa B DNA binding was detected after stimulation of HUVEC with NiCl2 or CoCl2. Supershift analysis using antisera against p50 and p65 confirmed the authenticity of the induced NF-kappa B complex. Neutralizing Abs against TNF-alpha and IL-1 did not inhibit metal hapten-induced activation of NF-kappa B, thus ruling out action via an indirect autocrine pathway. In addition, NiCl2-induced activation of NF-kappa B and adhesion molecule expression was inhibited by the antioxidant pyrrolidine dithiocarbamate, indicating the involvement of redox-dependent mechanisms. Furthermore, NiCl2 was found to induce dose-dependency mRNA production and protein secretion of the NF-kappa B-controlled proinflammatory cytokine IL-6. Our data suggest that distinct allergens represent a new class of so far unknown agents that induce NH-kappa B binding activity that subsequently modulates transcription of cytokine and adhesion molecule genes. Thus, pathomechanisms leading to contact hypersensitivity to NiCl2 and CoCl2 appear to involve not only Ag-specific Langerhans- and T cell-dependent events but also include direct effects on other immunocompetent cells such as the endothelium.

Migration inhibitory factor-related protein 8 (MRP8) and MRP14, two S-100-like Ca(2+)-binding proteins, have been described in cells of the epithelial lineage where they are either expressed constitutively (e.g. by mucosal squamous epithelium) or induced during disease (e.g. in keratinocytes during the course of psoriasis). Their biological function, however, is not yet clear. Recent studies have provided evidence that S-100-like proteins may interact with cytoskeletal components; we have therefore studied the biochemical properties and subcellular distribution of MRP8 and MRP14 in epithelial cells. TR146 human squamous carcinoma cells, which were found to express MRP8 and MRP14 in Northern and Western blot studies, were chosen for analysis. Cross-linking experiments using bis(sulphosuccinimidyl)suberate followed by SDS/PAGE and Western blot analysis revealed formation of heteromeric MRP8-MRP14 complexes. On subjecting TR146 cell lysates to two-dimensional gel electrophoresis and Western blotting, four distinct MRP14 isoforms could be identified resembling those described earlier in macrophages. A differential centrifugation technique revealed a Ca(2+)-dependent translocation of MRP8-MRP14 from the cytoplasm to the membrane and the Nonidet P40-insoluble cytoskeletal fraction. Double-label immunofluorescence microscopy of Ca2+ ionophore A23187-stimulated TR146 cells and cytochalasin B and demecolcine cytoskeleton disruption studies identified these structures as keratin intermediate filaments. Ca(2+)-dependent binding of MRP8-MRP14 to keratin filaments was additionally confirmed by an in vitro binding assay. In conclusion, our data suggest that MRP8 and MRP14 may be involved in Ca(2+)-dependent reorganization of cytoskeletal filaments in epithelial cells, which could be of importance for events associated with differentiation and inflammatory activation.

VCAM-1 (vascular cell adhesion molecule-1) is a cytokine-inducible adhesion molecule which is known to mediate adhesion of mononuclear cells to endothelial cells in vitro via binding to the integrin VLA-4 (very late antigen-4). To further elucidate the role and regulation of VCAM-1 in vivo, we compared in vitro and in vivo expression of VCAM-1 in response to cytokines and investigated immunohistochemically the expression of VCAM-1 in three murine models of experimental inflammation. These models differed with regard to the pathogenetic mechanism and the subsequent infiltrate: allergic contact dermatitis (ACD) to DNFB as a T cell-controlled, DTH type of inflammation, cutaneous infection with Leishmania major as a chronic granulomatous inflammation and the cauterized cornea as a model for acute inflammation. VCAM-1 was found to be markedly enhanced on vascular endothelia in all types of inflammation and after subcutaneous administration of LPS and TNF-alpha. Administration of IL-4, however, failed to induce VCAM-1 both in vivo and in vitro. The increased VCAM-1 expression in the inflammatory models correlated with the appearance of infiltrating monocytes/macrophages. A concomitant influx of CD4-positive/CD8-positive lymphocytes was only observed in ACD and Leishmaniasis.

MRP8 and MRP14, two S100-like calcium-binding proteins, are expressed during differentiation of monocytes/macrophages. Both assemble to different noncovalently associated complexes that are supposed to represent the biologically active states. The present study was intended to investigate the molecular basis of macrophage heterogeneity with respect to expression and complex formation of MRP8 and MRP14 during acute and chronic rejection of renal allografts. First, specificity of antisera and mAbs to be directed against MRP8, MRP14, or MRP8/MRP14 heterodimers was determined by immunocytochemical and Western blot analyses of L132 fibroblasts (co-)transfected with MRP8 and/or MRP14 cDNA. Then, immunohistochemical analysis of biopsy specimens obtained from kidney allografts after acute rejection was performed, revealing a parallel expression of MRP8 and MRP14 with coincident MRP8/MRP14 heterodimer formation in infiltrating monocytes. In contrast, chronic allograft rejection was characterized by a subpopulation of monocytes defined by the absence of MRP8/MRP14 complex formation despite expression of MRP8 and MRP14 monomers. Double-labeling experiments showed that this was due in part to differential expression of MRP8 and MRP14 in infiltrate macrophages of chronic rejection. The data presented demonstrate for the first time differences in MRP8/MRP14 complex assembly by infiltrating monocytes in situ. These seem to be of pathophysiological relevance since complex formation defines subpopulations of monocytes associated with distinct pathways of immunological reactions. Differences in the mode of calcium-dependent signaling may, therefore, be of importance for understanding the molecular basis of macrophage heterogeneity during acute and chronic allograft rejection.

MRP8 and MRP14 are two calcium-binding proteins of the S-100 family the expression of which is restricted to distinct stages of monocytic differentiation. Heteromeric MRP8/MRP14 complexes have been shown to represent their biologically active forms. However, it is not as yet clear whether biochemical modification of complexes, or regulation on the transcriptional level, are responsible for the control of MRP8/MRP14 expression. Employing Western-blot analysis and metabolic labelling we have demonstrated that patterns and metabolism of MRP8/MRP14 complexes do not change during up- or down-regulation of MRP8/MRP14. By Northern-blot analysis it was shown that MRP8/MRP14 are regulated at the transcriptional level rather than by biochemical modification of the complexes. Elevation of intracellular calcium levels by A23187, as well as by thapsigargin, was found to lead to specific down-regulation of MRP8/MRP14 mRNA which is in contrast with data reported for inflammatory factors such as interleukin-1 or tumour necrosis factor alpha. Concomitant application of actinomycin D and calcium ionophore indicated that this suppressive effect is mediated by decreased synthesis rather than increased degradation of MRP8/MRP14 mRNA. Finally, we demonstrated that calcium-mediated down-regulation of MRP8-MRP14 can be antagonized by cycloheximide, suggesting that a calcium-induced repressor protein is responsible for suppression of MRP8-MRP14 at the transcriptional level. Our data indicate that the function of MRP8-MRP14 is restricted to events associated with early stages of myelomonocytic activation.

Macrophages are supposed to play a key role in inflammatory and tumor angiogenesis. Their importance derives from (1) their ubiquitous presence in normal and especially inflamed tissues, (2) their potential to become activated in response to appropriate stimuli, and (3) their repertoire of secretory products. By release of proteases, growth factors (bFGF, GM-CSF, TGF-alpha, IGF-I, PDGF, VEGF/VPF, TGF-beta), and other monokines (IL-1, IL-6, IL-8, TNF-alpha, substance P, prostaglandins, interferons, thrombospondin 1), activated macrophages have the capability to influence each phase of the angiogenic process, such as alterations of the local extracellular matrix, induction of endothelial cells to migrate or proliferate, and inhibition of vascular growth with formation of differentiated capillaries. This review describes macrophage physiology and the influence of macrophage secretory products on the different phases of angiogenesis in vitro and in vivo.

The present study was initiated to identify the antigen recognized by the monoclonal antibody (MAb) MAC387 which is widely used for phenotypical characterization of myelomonocytic cells in situ. MAC387 has been described to show a similar reaction pattern as antisera to a complex formed by the calcium-binding proteins MRP8 and MRP14. However, the exact nature of the molecule recognized by MAC387 has been controversial. Using Western blot analysis, MAC387 was found to detect a single protein band of 14 kDa in lysates of human monocytes and granulocytes. Transfection of embryonic lung fibroblasts L132 with either MRP8 or MRP14 cDNA revealed that MAC387 reacts with MRP14 but not MRP8. This finding was confirmed by dot blot analysis of recombinant MRP8 and MRP14. Our data thus provide unequivocal evidence that MAC387 is a monoclonal antibody directed against the calcium-binding protein MRP14.

MRP8 and MRP14 are members of the S-100 family of Ca(2+)-binding proteins and are expressed by granulocytes and monocytes. Members of this family have been described to be involved in membrane and cytoskeleton interactions; we therefore studied the subcellular distribution of MRP8/MRP14 in cultured human monocytes at the ultrastructural level. Monospecific rabbit antisera against MRP8 and MRP14 and a monoclonal antibody (moAb 27E10), which exclusively recognizes the MRP8/MRP14 heterodimer but not the monomers, were used in both immunoperoxidase/preembedding- and immunogold/cryotechniques. Comparing non-stimulated monocytes with Ca2+ ionophore A23187-treated cells, we could demonstrate that MRP8 and MRP14 associate with membrane and cytoskeletal structures in a Ca(2+)-dependent manner. Employing moAb 27E10, MRP8/MRP14 complexes were shown to be translocated to these cellular components. In addition, immunogold double-labelling experiments revealed a clear co-localization of MRP8/MRP14 complexes with the type III intermediate filament vimentin. Analysis of immunogold-labelled cryosections of renal allografts after acute vascular rejection demonstrated that a subpopulation of infiltrating macrophages showed a similar association of MRP8/MRP14 to the cytoskeleton in situ; this finding emphasizes the in vivo relevance of our observations. We conclude that Ca(2+)-dependent translocation of MRP8/MRP14 occurs to distinct subcellular components suggesting a role of these proteins for the modulation of cytoskeletal and membrane interactions.

CsA, AZA, and dexamethasone (DEX) were examined for their ability to modulate expression of surface antigens on human monocytes. Peripheral blood monocytes were cultured for 1 day, treated with various concentrations of immunosuppressants, and subsequently analyzed for expression of macrophage differentiation antigen RM3/1, intercellular adhesion molecule-1 (CD54), beta 2-integrin (CD18), and HLA-DR using flow cytometry and indirect immunofluorescence microscopy. The RM3/1 antigen was found to be significantly down-regulated by CsA and AZA in a dose-dependent manner, whereas DEX led to a marked up-regulation of the RM3/1 molecule. In contrast, expression of CD54, CD18, and HLA-DR by macrophages was not affected by treatment with these immunosuppressants. Effects of AZA, CsA, and DEX on RM3/1 surface expression were shown to be mediated via modulation of RM3/1 de novo synthesis. Immunohistochemical analysis of rejected renal allografts revealed that the RM3/1 antigen is expressed by the majority of infiltrating macrophages. Our data demonstrate that CsA, AZA, and DEX differentially affect gene expression, resulting in distinct macrophage phenotypes. Characterization of the RM3/1+ macrophage population in vitro and during the course of allograft rejection in vivo may thus provide further insights regarding the mode of immunosuppressant action on nonspecific effector mechanisms.

The effects of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) on leukocyte infiltration during allergic contact dermatitis (ACD) in mice were studied. Concomitant topical application of SP or CGRP with the allergen oxazolone resulted in enhanced leukocyte recruitment at the sites of challenge. Immunohistochemical studies revealed that the numbers of T-helper (L3T4+) and cytotoxic (Lyt-2) lymphocytes and infiltrating macrophages (BM8+) were increased. In addition, ICAM-1 and MHC class II molecule expression by these cells was enhanced after neuropeptide application. Analysis by confocal laser scanning microscopy revealed an increase in the immunoreactivity for SP and CGRP in nerve fibres during the course of ACD. Flow cytometry studies showed that SP and CGRP did not upregulate expression of the adhesion molecules ICAM-1 and VCAM-1 by murine endothelial cell lines in vitro. This suggests that increased infiltration of leukocytes during ACD is not a consequence of direct neuropeptide-promoted upregulation of endothelial adhesion molecules in vivo. In conclusion, our observations provide evidence for a modulatory role of neuropeptides in the pathogenesis of ACD.

Resistance to murine leishmaniasis has been related to the propagation of specific Th cell subsets (Th1 and Th2). This study shows that there are differences between resistant and susceptible mice in the initial myelomonocytic infiltrate, which precede the specific T cell response. After subcutaneous injection of 2 x 10(7) Leishmania major into footpads of resistant C57Bl/6 and susceptible BALB/c mice we performed immunohistochemical studies on the infiltrate. Two days after infection the percentage of more mature, F4/80-positive macrophages in the lesion increased faster in C57Bl/6 mice (63%) than in BALB/c mice (29%). The same strain-specific differences were observed after infection of corresponding strains of athymic mice (57.2% in C57Bl/6 nu/nu; 33.6% in BALB/c nu/nu), thus excluding a T cell-controlled phenomenon. After 1 wk the infiltrate in susceptible mice began to reveal significantly more cells containing MRP14, which is expressed by granulocytes and less mature monocytes but not by mature macrophages. No corresponding differences were found between athymic strains, suggesting that at this point organization of the infiltrate falls under control of protective T cells. In bone marrow cultures of BALB/c and C57Bl/6 mice, the percentage of F4/80-positive macrophages was also increasing faster in C57Bl/6 mice than in BALB/c mice. Increased expression of the F4/80 Ag was associated with higher leishmanicidal activity of C57Bl/6 macrophages. MRP14-positive bone marrow cells on the other hand were rarely infected by parasites. We suggest 1) that the earlier appearance of leishmanicidal macrophages in lesions of C57Bl/6 mice could influence propagation of either Th1 or Th2 cells by reduction of parasite load or by differential secretion of decisive cytokines and 2) that the diffuse accumulation of granulocytes and inflammatory monocytes in susceptible mice facilitates spread of disease.

MRP8 and MRP14 are two Ca(2+)-binding proteins of the S-100 family expressed by myelomonocytic cells. Both proteins assemble to noncovalently associated complexes in a Ca(2+)-dependent manner. Members of the S-100 family are known to play a role in cytoskeletal-membrane interactions; therefore, we investigated the subcellular distribution of MRP8/MRP14 and their complexes in human monocytes. Using differential centrifugation and subsequent Western blot or enzyme-linked immunosorbent assay analysis, we found that MRP8/MRP14 were almost completely translocated from the cytoplasma to membrane and cytoskeletal structures in a Ca(2+)-dependent manner. Using a cross-linking technique, complexed forms of MRP8/MRP14 were found to be associated with the plasma membrane. Analysis of MRP-transfected L132 cells showed that the MRP8 as well as the MRP14 component of the MRP8/MRP14 complex may independently bind to membrane and cytoskeletal structures. Furthermore, immunogold electron microscopy showed a colocalization of MRP8/MRP14 and the intermediate filament type III protein vimentin in A23187-treated monocytes. Our data indicate that, in analogy to other S-100-like proteins, MRP8 and MRP14 play a role in Ca(2+)-dependent cytoskeletal-membrane interactions. Restriction of MRP8/MRP14 expression to distinct stages of myelomonocytic differentiation suggests that these proteins are involved in highly specific pathways of intracellular signaling in phagocytes.

Activation of vascular endothelium is a key event in the initial phase of an inflammatory reaction e.g. to contact allergens. In the present study the effect of two common contact sensitizers, NiCl2 and CoCl2, on endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1, E-selectin) was studied. NiCl2 and CoCl2 were found to upregulate these adhesion molecules on human umbilical vein endothelial cells (HUVEC) in a dose- and time-dependent manner as could be demonstrated by flow cytometry and enzyme-linked immunosorbent assay. During organ culture of foreskin samples treatment with NiCl2 led to upregulation of ELAM-1 and ICAM-1 but not VCAM-1 on microvascular endothelium. Induction of adhesion molecules by NiCl2 was found to depend on de novo mRNA and protein synthesis and could be blocked by the protein kinase inhibitor H-7 in a dose-dependent manner but not by HA-1004 and staurosporine. An autocrine IL-1-dependent mechanism mediating NiCl2 effects could be excluded. Our data demonstrate that allergens as NiCl2 and CoCl2 are capable of directly activating endothelium which is an important step in evolving inflammatory reactions and may thus be of relevance for the pathogenesis of contact hypersensitivity.

Intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1, E-selectin) are endothelial surface molecules that play a role for leukocyte recruitment to sites of inflammation, e.g., during contact hypersensitivity. We studied the effects of sensitizing agents (2,4-dinitro-benzenesulfonic acid, metal salt haptens) and chemically related substances on endothelial adhesion molecule expression. Using flow cytometry and an enzyme-linked immunosorbent assay, NiCl2 and, to a lesser extent, CoCl2 were found to up-regulate ICAM-1, VCAM-1, and ELAM-1 expression on cultured human umbilical vein endothelium whereas the other substances tested showed no effects. Induction of adhesion molecules by NiCl2 required de novo mRNA and protein synthesis. Up-regulation could be blocked by kinase inhibitor H-7 but not staurosporine, suggesting involvement of phosphorylation events independent of protein kinase C activation. Concomitant application of NiCl2 and neutralizing antibodies to IL-1 did not block up-regulation by the hapten demonstrating that the latter did not act via an IL-1-dependent autocrine mechanism. Regarding ELAM-1 induction, pre-treatment for 24 h with NiCl2 produced hyporesponsiveness to IL-1 and TNF-alpha upon restimulation, suggesting that NiCl2 and these cytokines may partially share a common pathway of activation. In addition, analysis of cultured foreskin specimens revealed that NiCl2 may induce up-regulation of ELAM-1 on microvascular endothelium in vivo. Our data demonstrate that both Ni++ and Co++ to which simultaneous contact sensitivity is frequently observed have the ability to directly up-regulate endothelial adhesion molecules. This shared property may represent an adjuvant mechanism that promotes sensitization and elicitation events in contact hypersensitivity to these haptens.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a cell-surface glycoprotein which has been shown to play an important role for cell/cell interaction. Little is known about its occurrence in the murine monocyte/macrophage (M phi) lineage; hence, we analyzed ICAM-1 expression in cells and cell lines representing different stages of M phi maturation and studied its regulation during inflammatory activation. Flow cytometric analysis of bone marrow-derived M phi cultured in the presence of M-CSF, of thioglycollate-elicited peritoneal M phi and of M1 myeloblasts differentiated by lipopolysaccharide (LPS) treatment revealed that ICAM-1 is increasingly expressed during monocytic maturation. Accordingly, the myelomonocytic cell lines RMB.TG, WEHI.TG, J774A and P388D, which can be ordered in a linear differentiation sequence, showed increasing levels of ICAM-1 expression. Furthermore, ICAM-1 expression by bone marrow-derived M phi could be up-regulated by tumor necrosis factor-alpha, interferon-gamma and LPS. In two models of murine experimental inflammation, i.e. induction phase of contact hypersensitivity and cutaneous leishmaniasis, which are both dependent on M phi/T cell interaction, M phi expressing ICAM-1 were found to be highly abundant. In addition, it was demonstrated that co-culture of Leishmania maior parasites with bone marrow M phi led to up-regulation of ICAM-1 on these cells. In conclusion, our data clearly demonstrate that ICAM-1 is increasingly being expressed during maturation of murine M phi. Cytokines and inflammatory stimuli modulate M phi ICAM-1 expression as well thus referring to its considerable role during inflammation, e.g. providing accessory or costimulatory signals for T cell activation.

MRP8 and MRP14 are two calcium-binding proteins of the S-100 family which are expressed during distinct stages of monocytic maturation. To further investigate their regulation the human leukemic cell line HL-60 which can be induced to differentiate to monocytes/macrophages by 12-O-tetradecanoylphorbol 13-acetate (TPA), 1,25-(OH)2 Vitamin D3 (VD3), tumor necrosis factor-alpha (TNF-alpha) and interferon-g (IFN-g) were analyzed for expression of MRP8/MRP14. Employing Northern blotting, a sandwich enzyme-linked immunosorbent assay and immunocytochemical analysis we determined MRP8/MRP14 mRNA and protein levels, which were found to be elevated after VD3 and reduced after TPA treatment. TNF-alpha and IFN-g did not affect MRP8/MRP14 levels. Western blot analysis revealed that formation of MRP8/MRP14 to biologically active complexes which has previously been shown to be a calcium-mediated process is not dependent on the differentiation stages of HL-60 cells. Restriction of MRP8/MRP14 expression to only distinct pathways of monocytic differentiation in HL-60 cells may thus reflect different functional phenotypes of monocytes/macrophages in vivo.

In the present study we analyzed expression and biochemical properties of the two recently cloned calcium-binding proteins MRP8 and MRP14, both members of the S-100 family, in murine myelomonocytic cells. Expression of MRPs was found to be restricted to granulocytes and distinct stages of macrophage differentiation when compared to the expression of other markers (Mac-1, F4/80) in transformed macrophage cell lines (M1, RMB.TG, WEHI.TG, J774A, P388D) and resident and exudate peritoneal and tissue macrophages. Similarly, mRNA and protein levels of MRP8/MRP14 in murine bone marrow cells decreased during culture in L cell-conditioned medium. Applying a cross-linking technique, the formation of noncovalently associated heteromeric MRP8/MRP14 complexes of 25, 35, and 48 kd was demonstrated. Our data suggest that MRP8/MRP14 expression and complexation are characteristic for granulocytes and distinct stages of macrophage differentiation in mice. Analysis of the MRP8/MRP14+ phenotype of macrophages may provide further insights into the mechanisms underlying macrophage differentiation.

Environmental influences are increasingly recognized as nonspecific modulators triggering and aggravating allergic diseases. To obtain better insight into the pathomechanisms of these nonantigen-specific phenomena, we studied the augmenting and inhibitory effects of croton oil and glucocorticosteroid (GC) on the induction of murine allergic contact dermatitis. Application of the irritant croton oil simultaneously with the sensitization dose of the contact allergen oxazolone strongly amplified the inflammatory reaction (measured as ear swelling) during the effector phase. Immunohistologically, this effect correlated with an increased percentage of MRP8- and MRP14-positive phagocytes in the infiltrate of the early inflammatory reaction 8 h after sensitization with oxazolone plus croton oil as compared to oxazolone alone (62 vs. 27%; p < 0.05). Intravenously administered GC was used as an inhibitor of contact sensitization. The suppressive effect of GC on sensitization was dependent on the time of its application: suppression of the inflammatory reaction during the effector phase was clearly more pronounced when GC had been injected 1 day as compared to 1 h before sensitization. Correspondingly, the percentage of BM8-positive macrophages in the infiltrate of the early inflammatory reaction 8 h after sensitization was differentially decreased depending on the time of GC application: suppression of the percentage of BM8-positive macrophages was clearly more pronounced when GC had been injected 1 day as compared to 1 h before sensitization (17 vs. 25%; base value without GC 35%; p < 0.05). Furthermore, the percentage of BM8-positive macrophages in the inflammatory infiltrate of the effector phase was increased when sensitization had been enhanced by concomitant irritant contact dermatitis (47 vs. 59%; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Monocytes/macrophages expressing an epitope recognized by a monoclonal antibody 27E10 are present in acute but are absent in chronic inflammatory disorders. This report shows that the 27E10 antigen is formed by noncovalent association of the two Ca(2+)-binding proteins MRP8 and MRP14 which belong to the S100 protein family. Identification has been confirmed immunochemically, by matrix-assisted UV-laser desorption/ionization spectrometry and by partial amino acid sequencing. Surface expression of the MRP8/MRP14 complex on a subset of monocytes is reported for the first time and shown to be up-regulated in a Ca(2+)-dependent manner. The 27E10 surface-positive monocytes isolated by cell separation techniques release high amounts of tumor necrosis factor-alpha and interleukin-1 beta in contrast to their 27E10 surface-negative counterparts thus emphasizing their role in inflammation.

The immune response to an allergen is not only dependent on the inflammatory stimulus, but also on the genetic disposition of the individual. Important effector cells in the immune response are myelomonocytic cells in their various differentiation stages. We recently described the expression of MRP8 and MRP14, two calcium-binding proteins of the S-100 family, by these cells during inflammatory activation. Here, we investigated whether their expression in murine contact dermatitis is dependent on the stimulus by which dermatitis is elicited, and if it is related to the genetic constitution of different inbred strains of mice. Therefore we performed immunohistochemical studies on the distribution of MRP8- and MRP14-positive cells during experimentally induced allergic (ACD) and irritant contact dermatitis (ICD). Both forms of dermatitis were elicited in BALB/c and C57B1/6 mice. BALB/c mice were found to react with a more intense inflammatory response in both ACD and ICD (high responders) than C57Bl/6 mice (low responders). The expression of MRP8 and MRP14 in both forms of dermatitis correlated with the early influx of macrophages and with the cell density of the infiltrate. Also the percentage of MRP8- and MRP14-positive cells in the infiltrate during ACD or ICD was higher in the more intense inflammatory reaction of BALB/c mice compared to C57Bl/6 mice. We conclude that MRP8 and MRP14 define a differentiation stage of inflammatory macrophages and that their expression correlates with the activity of inflammatory processes.

It is supposed that neuropeptides participate in the regulation of delayed-type hypersensitivity (DTH) reactions. However, their function in this kind of immune response is not known presently. Therefore, in vivo studies were initiated to test the effect on allergic (ACD) and irritant contact dermatitis (ICD) of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), and somatostatin (SOM), which are released from afferent neurons in the skin. Each neuropeptide was applied topically at the site of contact with the allergen (oxazolone) or irritant (croton oil) during the challenge and sensitization phase of contact dermatitis. The intensity of the inflammation was measured as an increase of ear-swelling response, which represents the degree of plasma extravasation in the early phase of inflammation. Neuropeptides alone led only to a distinct vasodilation. All three neuropeptides were equally able to increase allergic and irritant inflammation. Even minor irritant stimuli were enhanced. Beyond that, CGRP was able to boost sensitization, whereas SOM and SP did not show any effects on the sensitization process. The results presented demonstrate that neuropeptides increase plasma extravasation independent of the pathogenesis of inflammation and may act as priming substances for other mediators of increased vascular permeability. In addition, CGRP enhances the sensitization process.

Irritant contact dermatitis to croton oil in BALB/cByJ, C57Bl/6J and six recombinant inbred CxB strains of mice was investigated in relation to variations in endothelial migration inhibitory factor (MIF) reactivity. MIF has been shown to be a mediator of cellular immunity and operates as a differentiation signal inducing an inflammatory type of macrophage. The intensity of the ear swelling response reached a maximum 8 h after induction of contact dermatitis, with highest values in BALB/cByJ and CxB4/ByAH mice and weak reactions in CxB2/ByAE, CxB7/ByAK, C57Bl/6J and CxB1/ByAD mice. After the same time period (8 h) cryostat sections were immunostained for capillary endothelium expressing MIF. The most pronounced MIF expression was observed in BALB/cByJ mice, and CxB4/ByAH mice showed intermediate reactions and the other strains weak reactions. Endothelial MIF expression correlated well with the intensity of ear swelling (Pearson's correlation coefficient 0.82). Patterns of endothelial MIF expression in recombinant inbred strains suggest that endothelial MIF expression is not under the control of a single gene. Our data support the hypothesis that endothelial MIF expression plays a prominent role in inflammatory events and correlates with the severity of inflammation.

A majority of angiogenic factors has been shown to be produced by macrophages. This review will give a concise description of their biochemical nature, their isolation from macrophages and their angiogenic activity. Among the factors with mitogenic effects on endothelial cells are basic fibroblast growth factor (bFGF), transforming growth factor-alpha (TGF-alpha) and very probably insulin-like growth factor-1 (IGF-1). Other secretory products such as angiotropin and human angiogenic factor (HAF) are nonmitogenic but promote angiogenesis by inducing migration of endothelial cells. Prostaglandins, platelet-derived growth factor (PDGF), granulocyte-macrophage- and granulocyte-colony stimulating factor (GM-CSF, G-CSF), interleukin 6 (IL-6) and angiotensin converting enzyme (ACE) have also been shown to be angiogenic, but their mode of action is still to be clearly defined. As the extracellular matrix appears to be involved in the control of angiogenesis, macrophage-derived factors that can alter this structure via degradation or via the clotting system will also be discussed. Tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and transforming growth factor-beta (TGF-beta) have complex actions on endothelial cells, and can partially inhibit angiogenesis. Among the factors which solely inhibit neovascularization are the interferons. As it is not known whether all of these factors play a role in angiogenesis in vivo attempts to detect them in situ during the course of neovascularization will be described. Finally macrophages will be discussed as cells that may not be mandatory for each phase of the angiogenic process but whose angiogenic capabilities are comprehensive and unsurpassed by any other cell.

Two strains of mice, BALB/c and C57Bl/6, which are known to differ in their inflammatory responsiveness to allergens, were analyzed regarding their expression of macrophage migration inhibitory factor (MIF). Allergic contact dermatitis to 2,4-dinitro-1-fluorobenzene and irritant contact dermatitis to croton oil were studied immunohistologically at designated time intervals after elicitation. BALB/c mice presented a significantly more intense ear swelling response than C57Bl/6 mice and showed a strong endothelial MIF expression in the early phase of inflammation in both allergic and irritant contact dermatitis. Endothelial MIF expression was much weaker in C57Bl/6 mice. Furthermore, the infiltration rate of inflammatory cells (MIF+ and BM8+ macrophages, Lyt2+ and L3T4+ T cells) was significantly higher in BALB/c than in C57Bl/6 mice. We conclude that genetically determined differences of susceptibility to allergens and irritants in BALB/c and C57Bl/6 mice are reflected by the intensity of MIF expression in the microvascular endothelium and immigrating inflammatory cells. MIF seems to appear as first molecular equivalent of developing inflammation and probably determines the degree of cellular infiltration.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the interaction of immunocompetent cells in inflammation. It contributes to lymphocyte recruitment, antigen presentation and T-cell activation. Recently, the murine homologue of ICAM-1, originally designated as MALA-2, has been identified. The monoclonal antibody YN1/1.7 for murine ICAM-1 enables the analysis of ICAM-1 expression in murine allergic contact dermatitis, a prototype of cutaneous T-cell-mediated inflammatory response. At 0, 1, 8, 24, 48, 72 h, and 7 and 14 days after challenge with 2,4-dinitrofluoro-1-benzene (DNFB) cryostat sections of ear skin were immunostained for ICAM-1, I-A and mononuclear cells (L3T4, Lyt-2, BM8). In normal skin ICAM-1 labeling was restricted to endothelial cells and dermal dendritic cells/macrophages; keratinocytes (KCs) did not express ICAM-1. The dermal cellular infiltrate increased progressively from 8 to 72 h after DNFB challenge. The majority of infiltrating cells were BM8+ macrophages (75%) and L3T4+ (10%) or Lyt-2+ T cells (10%); maximally 30% of those stained positive for ICAM-1. At 24 h, focal ICAM-1 expression on KCs developed, reached a maximum at 72 h and faded thereafter. Migration of T cells into the epidermal layer started at 48 h at sites which had already expressed ICAM-1. Our data provide evidence that expression of ICAM-1 by epidermal cells precedes infiltration of the epidermis by T lymphocytes as shown before in human cutaneous disorders. Thus, a mouse model may be useful to investigate the role of ICAM-1 in inflammation further.