Potential neurotoxic activity of diverse molecules released by astrocytes.

Astrocytes are the main support cells of the central nervous system. They also participate in neuroimmune reactions. In response to pathological and immune stimuli, astrocytes transform to reactive states characterized by increased release of inflammatory mediators. Some of these molecules are neuroprotective and inflammation resolving while others, including reactive oxygen species (ROS), nitric oxide (NO), matrix metalloproteinase (MMP)- 9, L-glutamate, and tumor necrosis factor α (TNF), are well-established toxins known to cause damage to surrounding cells and tissues. We hypothesized that similar to microglia, the brain immune cells, reactive astrocytes can release a broader set of diverse molecules that are potentially neurotoxic. A literature search was conducted to identify such molecules using the following two criteria: 1) evidence of their expression and secretion by astrocytes and 2) direct neurotoxic action. This review describes 14 structurally diverse molecules as less-established astrocyte neurotoxins, including C-X-C motif chemokine ligand (CXCL)10, CXCL12/CXCL12(5-67), FS-7-associated surface antigen ligand (FasL), macrophage inflammatory protein (MIP)- 2α, TNF-related apoptosis inducing ligand (TRAIL), pro-nerve growth factor (proNGF), pro-brain-derived neurotrophic factor (proBDNF), chondroitin sulfate proteoglycans (CSPGs), cathepsin (Cat)B, group IIA secretory phospholipase A2 (sPLA2-IIA), amyloid beta peptides (Aß), high mobility group box (HMGB)1, ceramides, and lipocalin (LCN)2. For some of these molecules, further studies are required to establish either their direct neurotoxic effects or the full spectrum of stimuli that induce their release by astrocytes. Only limited studies with human-derived astrocytes and neurons are available for most of these potential neurotoxins, which is a knowledge gap that should be addressed in the future. We also summarize available evidence of the role these molecules play in select neuropathologies where reactive astrocytes are a key feature. A comprehensive understanding of the full spectrum of neurotoxins released by reactive astrocytes is key to understanding neuroinflammatory diseases characterized by the adverse activation of these cells and may guide the development of novel treatment strategies.

CCR1-mediated STAT3 tyrosine phosphorylation and CXCL8 expression in THP-1 macrophage-like cells involve pertussis toxin-insensitive Gα(14/16) signaling and IL-6 release.

Agonists of CCR1 contribute to hypersensitivity reactions and atherosclerotic lesions, possibly via the regulation of the transcription factor STAT3. CCR1 was demonstrated to use pertussis toxin-insensitive Gα(14/16) to stimulate phospholipase Cß and NF-κB, whereas both Gα(14) and Gα(16) are also capable of activating STAT3. The coexpression of CCR1 and Gα(14/16) in human THP-1 macrophage-like cells suggests that CCR1 may use Gα(14/16) to induce STAT3 activation. In this study, we demonstrated that a CCR1 agonist, leukotactin-1 (CCL15), could indeed stimulate STAT3 Tyr(705) and Ser(727) phosphorylation via pertussis toxin-insensitive G proteins in PMA-differentiated THP-1 cells, human erythroleukemia cells, and HEK293 cells overexpressing CCR1 and Gα(14/16). The STAT3 Tyr(705) and Ser(727) phosphorylations were independent of each other and temporally distinct. Subcellular fractionation and confocal microscopy illustrated that Tyr(705)-phosphorylated STAT3 translocated to the nucleus, whereas Ser(727)-phosphorylated STAT3 was retained in the cytosol after CCR1/Gα(14) activation. CCL15 was capable of inducing IL-6 and IL-8 (CXCL8) production in both THP-1 macrophage-like cells and HEK293 cells overexpressing CCR1 and Gα(14/16). Neutralizing Ab to IL-6 inhibited CCL15-mediated STAT3 Tyr(705) phosphorylation, whereas inhibition of STAT3 activity abolished CCL15-activated CXCL8 release. The ability of CCR1 to signal through Gα(14/16) provides a linkage for CCL15 to regulate IL-6/STAT3-signaling cascades, leading to expression of CXCL8, a cytokine that is involved in inflammation and the rupture of atherosclerotic plaque.

Leukotactin-1/CCL15 induces cell migration and differentiation of human eosinophilic leukemia EoL-1 cells through PKCdelta activation.

Leukotactin-1 (Lkn-1)/CCL15 is a CC chemokine that binds to the CCR1 and CCR3. Lkn-1 functions as an essential factor in the migration of monocytes, lymphocytes, and neutrophils. Although eosinophils express both receptors, the role of Lkn-1 in immature eosinophils remains to be elucidated. In this present study, we investigated the contribution of the CCR1-binding chemokines to chemotactic activity and in the differentiation in the human eosinophilic leukemia cell line EoL-1. Lkn-1 induced the stronger migration of EoL-1 cells than other CCR1-binding chemokines such as RANTES/CCL5, MIP-1alpha/CCL3 and HCC-4/CCL16. Lkn-1-induced chemotaxis was inhibited by pertussis toxin, an inhibitor of G(i)/G(o) protein; U73122, an inhibitor of phospholipase C and rottlerin, an inhibitor of protein kinase C delta (PKCdelta). Lkn-1 increased PKCdelta activity, which was partially blocked by the pertussis toxin and U73122. Lkn-1 enhanced the butyric acid-induced differentiation via PKCdelta after binding to the increased CCR1 because Lkn-1 caused EoL-1 cells to change morphologically into mature eosinophil-like cells. Likewise, Lkn-1 increased the expression of both eosinophil peroxidase (EPO) and the major basic protein (MBP). PKCdelta activation due to Lkn-1 is involved in migration, as well as the butyric acid-induced differentiation. This finding contributes to an understanding of CC chemokines in eosinophil biology and to the development of novel therapies for the treatment of eosinophilic disorders. This study suggests the pivotal roles of Lkn-1 in the regulation of the movement and development of eosinophils.

Human LZIP induces monocyte CC chemokine receptor 2 expression leading to enhancement of monocyte chemoattractant protein 1/CCL2-induced cell migration.

Chemokines and chemokine receptors play a role in migration of circulating leukocytes to the region of inflammation. Human LZIP is an uncharacterized transcription factor and is known to participate in leukotactin (Lkn)-1/CCL15-induced cell migration. We investigated the role of human LZIP in expression of CC chemokine receptors (CCRs) and its involvement in monocyte migration. RNase protection analysis showed that LZIP increased mRNA expression of CCR2 and CCR1 in THP-1 cells. Surface expressions of both CCR2 and CCR1 were also increased by LZIP. Results from an electrophoretic mobility shift assay showed that LZIP binds to the C/EBP element in the CCR2 promoter. LZIP also enhanced the chemotactic activities of monocyte chemoattractant protein-1/CCL2 and Lkn-1. These results suggest that LZIP regulates expression of chemokine receptors that are involved in monocyte migration.

Ocular surface expression and in vitro activity of antimicrobial peptides.

PURPOSE: Human ocular surface epithelia express four antimicrobial peptides (APs): beta -defensin (hBD) 1-3 and LL-37. Here the expression of additional APs (hBD 4-6, HE2beta 1; histatin-1, -3; liver expressed antimicrobial peptide-1, -2; macrophage inflammatory protein (MIP)-3alpha, and thymosin (T)beta -4) was sought and activity against common ocular pathogens studied. METHODS: AP expression was determined in human corneal and conjunctival epithelial cells (HCEC, HCjEC) by RT-PCR and in corneal sections by immunostaining. Antimicrobial assays were performed to assess peptide (hBD 1-3, LL-37, MIP-3alpha, and Tbeta 4) activity against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Staphylococcus epidermidis (SE) in the presence of NaCl or tears. RESULTS: HCEC and HCjEC expressed MIP-3alpha and Tbeta 4. hBD 1-3, MIP-3alpha, and Tbeta 4 showed activity against PA. hBD-3 had potent activity against SA and SE, whereas hBD-2, MIP-3alpha and Tbeta 4 had moderate activity and hBD-1 had none. NaCl markedly attenuated, and tears almost completely inhibited the activity of hBD 1-2 and Tbeta 4, but not that of hBD-3. CONCLUSIONS: The ocular surface epithelia additionally express MIP-3alpha and Tbeta 4 which have moderate antimicrobial activity. The current data support a role for hBD-3 as an antimicrobial peptide in vivo, but call in to question the effectiveness of some other APs. However, further study is required to conclusively elucidate the physiological role of each AP.

Macrophage-inflammatory protein-3alpha mediates epidermal growth factor receptor transactivation and ERK1/2 MAPK signaling in Caco-2 colonic epithelial cells via metalloproteinase-dependent release of amphiregulin.

Previously, we reported that normal colonocytes produce the memory CD4(+) T cell-directed chemokine MIP-3alpha, and that epithelial MIP-3alpha levels are elevated in inflammatory bowel disease. Interestingly, the unique receptor for MIP-3alpha, CCR6, is expressed by a variety of cell types including colonocytes, suggesting that MIP-3alpha may regulate additional biological activities in the intestine. The aim of this study was to determine whether MIP-3alpha can induce intestinal epithelial cell proliferation and to examine the signaling mechanisms that mediate this response. We show that nonstimulated Caco-2 and HT-29 colonic epithelial cells express CCR6, and that stimulation of Caco-2 cells by MIP-3alpha can dose dependently increase cell proliferation as well as activate the epidermal growth factor receptor (EGFR) and ERK1/2 MAPK. MIP-3alpha-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3alpha-mediated, but not EGF-mediated Caco-2 cell proliferation. Taken together, our findings indicate that MIP-3alpha can regulate mitogenic signaling in colonic epithelial cells and thus may serve an important homeostatic function in the intestine by regulating tissue turnover and maintenance of the epithelium, in addition to its role in regulating leukocyte recruitment.

Cryptotanshinone inhibits macrophage migration by impeding F-actin polymerization and filopodia extension.

We evaluated the anti-inflammatory effects of cryptotanshinone and tanshinone IIA, two major tanshinones isolated from Salvia miltiorrhiza, on chemoattractant-induced cell migration in RAW264.7 macrophages. Results showed that cryptotanshinone inhibited cell migration toward complement 5a (C5a) and macrophage inflammatory protein-1alpha (MIP-1alpha) in a concentration-dependent manner. In contrast, tanshinone IIA displayed less or even no effect on cell migration evoked by these chemoattractants. Both C5a- and MIP-1alpha-induced migration were clearly inhibited by cytochalasin B (an inhibitor of actin polymerization), but not by colchicine (an inhibitor of microtubule polymerization). Fluorescence staining demonstrated that cryptotanshinone as well as cytochalasin B, effectively reversed cell polarization and filopodia extension induced by both chemoattractants. Furthermore, C5a-evoked increase in F-actin fluorescence intensity was significantly suppressed by cryptotanshinone. Based on these observations, we suggest that cryptotanshinone exerts anti-migrating activity possibly by impeding F-actin polymerization and filopodia formation.

Cryptotanshinone inhibits chemotactic migration in macrophages through negative regulation of the PI3K signaling pathway.

BACKGROUND AND PURPOSE: Cryptotanshinone, the major tanshinone isolated from Salvia miltiorrhiza Bunge, exhibits anti-inflammatory activity. However, there is no report on the effect of cryptotanshinone on recruitment of leukocytes to inflammatory sites. We therefore assessed the effects of cryptotanshinone on macrophage chemotaxis. EXPERIMENTAL APPROACH: Macrophage migration induced by complement 5a (C5a) or macrophage inflammatory protein-1alpha (MIP-1alpha) was measured in vitro. Intracellular kinase translocation and phosphorylation was assessed by Western blotting. KEY RESULTS: RAW264.7 cell migration towards C5a (1 microg ml(-1)) was significantly inhibited by cryptotanshinone (1, 3, 10 and 30 microM) in a concentration-dependent manner. Primary human macrophages stimulated by C5a were similarly inhibited. C5a-evoked migration in RAW264.7 cells was significantly suppressed by wortmannin (phosphatidylinositol 3-kinase (PI3K) inhibitor), PD98059 (MEK1/2 inhibitor) and SB203580 (p38 mitogen-activated protein kinase (MAPK) inhibitor), but not by SP600125 (c-Jun N-terminal kinase (JNK) inhibitor), suggesting that activation of PI3K, ERK1/2 and p38 MAPK signal pathways was involved in responses to C5a. Western blotting revealed that cryptotanshinone significantly inhibited PI3K-p110gamma membrane translocation and phosphorylation of Akt (PI3K downstream effector protein) and ERK1/2 induced by C5a. However, neither p38 MAPK nor JNK phosphorylation was affected by cryptotanshinone. Wortmannin significantly attenuated C5a-induced PI3K-p110gamma translocation, Akt and ERK1/2 phosphorylation. PD98059 suppressed ERK1/2 phosphorylation but failed to modify PI3K-p110gamma translocation by C5a stimulation. Furthermore, MIP-1alpha-induced cell migration and PI3K-p110gamma translocation were also inhibited by cryptotanshinone in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: Inhibition of macrophage migration by cryptotanshinone involved inhibition of PI3K activation with consequent reduction of phosphorylation of Akt and ERK1/2.

Comparison of cellular functionality of human mesenchymal stromal cells and PBMC.

BACKGROUND: Human mesenchymal stromal cells (MSC) and PBMC play significant roles in repair processes following inflammation. Mechanisms of recruitment are still under investigation. METHODS AND RESULTS: MIP-1alpha induced the chemotactic migration of MSC but not of PBMC. Correlating with this, 7.7% of MSC expressed the chemokine receptor CCR-1, as shown by FACS analysis. In contrast, PBMC did not express CCR-1 or CCR-2 but did express CXCR-4 (81.9%) and CCR-7 (42.2%). Setum induced the chemotaxis of both cell types, and zymosan activation increased the migration of PBMC but not of MSC. Corresponding with this, C5a induced the migration of PBMC but not of MSC. Dose-dependent and -specific adhesion to fibronectin, fibrinogen, collagen type I and collagen type II could be demonstrated for MSC; in contrast, PBMC did not adhere to any of the investigated proteins. Real-time PCR of receptor expression revealed a 12.2-fold higher expression of alphav in MSC compared with PBMC. Incubation of MSC with tumor necrosis factor-alpha (TNFalpha) induced NFkappaB activation and increased the chemotactic response to serum and adhesion to fibronedtin. DISCUSSION: Chemotaxis and adhesion are crucial and differing cell fundtons of MSC and PBMC.

Role of human LZIP in differential activation of the NF-kappaB pathway that is induced by CCR1-dependent chemokines.

Human leucine zipper protein (LZIP) associates with CC chemokine receptor 1 (CCR1) and this protein-protein interaction should play an important role in leukocyte cell mobility. LZIP is known to regulate leukotactin-1 (Lkn-1)-dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1. Since Lkn-1 is engaged in the transcriptional activation of nuclear factor kappaB (NF-kappaB) and subsequent activation of the chemoattractant ability of leukocytes, we investigated the regulatory role of LZIP in the NF-kappaB pathway that is induced by CCR1-dependent chemokines. LZIP increased NF-kappaB-dependent luciferase activity in response to Lkn-1 in HOS/CCR1 cells and THP-1 cells. However, the NF-kappaB-dependent luciferase activities induced by other CCR1-dependent chemokines were not affected by LZIP overexpression. LZIP also increased Lkn-1-induced chemotactic activity through activation of the NF-kappaB pathway, whereas LZIP did not affect either the transactivation of NF-kappaB or the chemotactic activities induced by other CCR1-dependent chemokines. Western blot analysis showed that LZIP increased the degradation of IkappaBalpha induced by Lkn-1 but not by other CCR1-dependent chemokines. Results from electrophoretic mobility shift assay (EMSA) showed that LZIP enhanced the Lkn-1-induced DNA-binding activity of NF-kappaB. These data indicate that LZIP functions as a positive regulator in the NF-kappaB activation pathway that is triggered by Lkn-1 without affecting the transcriptional activation of NF-kappaB induced by other CCR1-dependent chemokines.

Role of CCR1 and CCR5 in homing and growth of multiple myeloma and in the development of osteolytic lesions: a study in the 5TMM model.

Multiple myeloma (MM) is a plasma cell malignancy, characterized by the localization of the MM cells in the bone marrow (BM), where they proliferate and induce osteolysis. The MM cells first need to home or migrate to the BM to receive necessary survival signals. In this work, we studied the role of CCR1 and CCR5, two known chemokine receptors, in both chemotaxis and osteolysis in the experimental 5TMM mouse model. A CCR1-specific (BX471) and a CCR5-specific (TAK779) antagonist were used to identify the function of both receptors. We could detect by RT-PCR and flow cytometric analyses the expression of both CCR1 and CCR5 on the cells and their major ligand, macrophage inflammatory protein 1alpha (MIP1alpha) could be detected by ELISA. In vitro migration assays showed that MIP1alpha induced a 2-fold increase in migration of 5TMM cells, which could only be blocked by TAK779. In vivo homing kinetics showed a 30% inhibition in BM homing when 5TMM cells were pre-treated with TAK779. We found, in vitro, that both inhibitors were able to reduce osteoclastogenesis and osteoclastic resorption. In vivo end-term treatment of 5T2MM mice with BX471 resulted in a reduction of the osteolytic lesions by 40%; while TAK779 treatment led to a 20% decrease in lesions. Furthermore, assessment of the microvessel density demonstrated a role for both receptors in MM induced angiogenesis. These data demonstrate the differential role of CCR1 and CCR5 in MM chemotaxis and MM associated osteolysis and angiogenesis.

Neutrophil recruitment in immunized mice depends on MIP-2 inducing the sequential release of MIP-1alpha, TNF-alpha and LTB(4).

Neutrophils are thought to play an important role in the tissue damage observed in various autoimmune diseases. Chemokines, cytokines and leukotrienes have recognized roles in the orchestration of neutrophil migration. We have recently shown that antigen-induced neutrophil migration into the peritoneum of immunized mice is mediated by macrophage-inflammatory protein (MIP)-1alpha which interacts with CCR1 and induces the sequential release of TNF-alpha and leukotriene B(4) (LTB(4)). The present study investigates the role of MIP-2 and CXCR2 in the cascade of events leading to mediator generation and neutrophil influx. Antigen challenge of immunized mice induced the expression of CXCR2 and the production of KC and MIP-2 proteins. Antigen-induced neutrophil migration was inhibited by a CXCR2 receptor antagonist (repertaxin) or an anti-MIP-2 antibody, but not by an anti-KC antibody. Administration of MIP-2 promoted a dose-dependent neutrophil migration in naive mice which was inhibited by repertaxin, anti-TNF-alpha, anti-MIP-1alpha antibodies or by MK886 (leukotriene synthesis inhibitor). MIP-2 administration induced the release of MIP-1alpha, TNF-alpha and LTB(4), and the release of the latter two was inhibited by anti-MIP-1alpha antibody treatment. Our studies highlight the intricate balance between mediator production and action during an immune-mediated inflammatory response and suggest a mediator cascade leading to neutrophil influx following antigen challenge of immunized mice: MIP-2 --> MIP-1alpha --> TNF-alpha --> LTB(4).

Interleukin-1beta-induced growth enhancement of Staphylococcus aureus occurs in biofilm but not planktonic cultures.

Staphylococcus aureus causes recalcitrant infections and forms resistant biofilms. Mechanisms of biofilm resistance to host defenses may include changes in gene expression that confer responsiveness to chemical mediators. In earlier studies fresh clinical isolates responded to inflammatory cytokines, but responsiveness was lost after multiple in vitro passages [Meduri et al. Cytokines IL-1beta, IL-6, and TNF-alpha enhance the In vitro growth of bacteria. Am J Respir Crit Care Med 1999;160:961-7]. Since biofilms more closely resemble in vivo growth and are implicated in recalcitrant infections, we hypothesized that biofilms, but not planktonic cells, would respond to cytokines. Biofilms were induced by ethanol in S. aureus ATCC 12600. Biofilms treated with 2 ng/mL interleukin-1beta (IL-1beta) for 6 h contained 2.5-fold more cells than untreated biofilms, but no growth-enhancement occurred in planktonic cultures. As determined by flow cytometry, IL-beta bound to 63.1% of biofilm cells, but only 11.2% of planktonic cells. Our results provide evidence of a differential response of biofilm and planktonic bacteria to chemical mediators, and suggest that biofilm bacteria may evade host defenses by growing more rapidly in response to the inflammatory mediators released by activated host defense cells.

Chemokine receptor CCR6 as a prognostic factor after hepatic resection for hepatocellular carcinoma.

BACKGROUND AND AIMS: Chemokines and their receptors have recently been shown to have major roles in cancer metastasis. The aim of this study was to determine whether the interaction between chemokine receptor 6 (CCR6) and its ligand, macrophage inflammatory protein-3 alpha (MIP-3alpha), correlates with metastasis of hepatocellular carcinoma (HCC). METHODS: To observe the reaction of CCR6 expressed cancer cells to MIP-3alpha stimulation, chemotactic and actin polymerization assays for both CCR6 high cells (HepG2) and CCR6 low cells (MCF-7) were performed. CCR6 mRNA levels in tumor specimens from 30 HCC patients were quantified by real-time polymerase chain reaction. Patients were classified into two groups, high (>or= 20 copies; n=10) CCR6 and low (<20 copies; n=20) CCR6 on the basis of CCR6 expression, and the groups were compared with respect to clinicopathological features. RESULTS: When HepG2 cells (CCR6 high) were stimulated with MIP-3alpha, they migrated in a dose-dependent manner, and formation of pseudopodia was observed. These phenomena were not observed in the CCR6 low cells. The incidence of intrahepatic metastasis was higher in the high CCR6 expression group than in the low CCR6 expression group (P<0.05). Disease-free survival was significantly poorer in the high CCR6 expression group than in the low CCR6 expression group (P<0.05). CONCLUSIONS: It was indicated that CCR6 might be associated with intrahepatic metastasis of HCC and might be able to become one of the prognostic factor after hepatic resection for HCC.

The Src kinase Lyn is required for CCR5 signaling in response to MIP-1beta and R5 HIV-1 gp120 in human macrophages.

CCR5 is a receptor for several beta chemokines and the entry coreceptor used by macrophage-tropic (R5) strains of HIV-1. In addition to supporting viral entry, CCR5 ligation by the HIV-1 envelope glycoprotein 120 (gp120) can activate intracellular signals in macrophages and trigger inflammatory mediator release. Using a combination of in vitro kinase assay, Western blotting for phospho-specific proteins, pharmacologic inhibition, CCR5 knockout (CCR5Delta32) cells, and kinase-specific blocking peptide, we show for the first time that signaling through CCR5 in primary human macrophages is linked to the Src kinase Lyn. Stimulation of human monocyte-derived macrophages with either HIV-1 gp120 or MIP-1beta results in the CCR5-mediated activation of Lyn and the concomitant Lyn-dependent activation of the mitogen-activated protein (MAP) kinase ERK-1/2. Furthermore, activation of the CCR5/Lyn/ERK-1/2 pathway is responsible for gp120-triggered production of TNF-alpha by macrophages, which is believed to contribute to HIV-1 pathogenesis. Thus, Lyn kinase may play an important role both in normal CCR5 function in macrophages and in AIDS pathogenesis in syndromes such as AIDS dementia where HIV-1 gp120 contributes to inappropriate macrophage activation, mediator production, and secondary injury.

Macrophage inflammatory protein (MIP)1alpha and MIP1beta differentially regulate release of inflammatory cytokines and generation of tumoricidal monocytes in malignancy.

The C-C chemokines, macrophage inflammatory protein (MIP)1alpha and MIP1beta are potent chemoattractants for the monocytes, which form an important component of the stroma of tumor tissue and may regulate tumor growth and associated inflammation. We examined the role of MIP1alpha and MIP1beta in inducing the release of inflammatory cytokines and the generation of tumoricidal monocytes from the peripheral blood monocytes (PBM) of healthy women and patients with carcinoma of breast (CaBr). Interleukin-1 (IL-1) and tumor necrosis factor (TNF) alpha release by the PBM was markedly stimulated by MIP1alpha in CaBr patients, but only marginally so in healthy women. In contrast, MIP1beta stimulated the release of these cytokines by the PBM of healthy women, but failed to do so in CaBr patients. MIP1alpha, but not MIP1beta, synergized with LPS in inducing the release of IL-1 from the PBM of both healthy women and CaBr patients. Both MIP1alpha and MIP1beta augmented respiratory bursts in PBM and generated tumoricidal PBM that killed T24 cells, MIP1alpha being more effective in CaBr patients and MIP1beta in healthy women. IFN-gamma co-stimulated and IL-4 suppressed MIP1alpha and beta-induced cytotoxicity in PBM. The synergy of IFN-gamma was more marked with MIP1alpha than with MIP1beta. The differential effects of MIP1alpha and MIP1beta on the PBM of healthy women and CaBr patients co-related with the levels of expression of CCR1 and CCR5 in these monocytes. The expression of CCR5 was higher than that of CCR1 in the PBM of healthy women and the PBM of the CaBr patients showed overexpression of CCR1 and downregulation of CCR5.

Tumor necrosis factor-alpha- and IL-4-independent development of Langerhans cell-like dendritic cells from M-CSF-conditioned precursors.

GM-CSF and transforming growth factor beta (TGFbeta ) are required for the generation of Langerhans cells (LC), members of the dendritic cell (DC) family. Tumor necrosis factor alpha (TNFalpha) and IL-4 can enhance LC differentiation from human monocytes or CD34(+) progenitors. Here, we show that M-CSF-cultured DC precursors derived from CD34(+) progenitors resemble dermal CD14(+) cells and readily convert to LC-like DC in GM-CSF/TGFbeta. The cells express Langerin, CD1a, and CCR6, migrate in response to CCR6 ligand CCL20, and contain Birbeck granules. TNFalpha and IL-4, added separately or together, have an inhibitory effect on LC differentiation. Cells differentiated in the presence of IL-4 and TNFalpha express low levels of CCR7. This suggests that M-CSF-conditioned DC precursors retain the capacity to efficiently undergo a differentiation program, giving rise to LC-like DC solely through the effect of GM-CSF and TGFbeta.

CXCL12 and CXCR4 expression by human gingival fibroblasts in periodontal disease.

CXCL12 is a CXC chemokine that is related to lymphocyte infiltration and angiogenesis in inflammatory sites such as arthritis. However, the expression and roles of CXCL12 in periodontal disease are uncertain. The aim of this study was to assess the expression of CXCL12 and its receptor, CXCR4, in periodontal tissue and to investigate the properties of CXCL12 and CXCR4 expression by human gingival fibroblasts (HGF). RT-PCR analysis revealed that CXCL12 and CXCR4 mRNA were expressed in both normal gingival tissues and periodontal diseased tissues. Immunohistochemistry disclosed that CXCL12 was expressed and CXCR4 positive cells were found in both normal and periodontal diseased gingival tissues. Our in vitro experiments elucidated that HGF constitutively produced CXCL12, and the levels were enhanced by stimulation with tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), regulated upon activation normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein 3(alpha) (MIP-3(alpha)). On the other hand, heat killed Porphyromonas gingivalis (P. gingivalis) and P. gingivalis LPS reduced the CXCL12 production by HGF. Flow cytometry analysis clarified that CXCR4 was highly expressed on HGF, and CXCR4 expression was abrogated by TNF-alpha, IFN-gamma and P. gingivalis LPS. Moreover, CXCL12 induced vascular endothelial growth factor (VEGF) production by HGF. Our results demonstrated that CXCL12 might be related to CXCR4+ cells infiltration and angiogenesis both in normal periodontal tissues and periodontal diseased tissue. P. gingivalis, a known periodontal pathogen, inhibits the production of CXCL12 and the expression of CXCR4 by HGF. This fact means that P. gingivalis may inhibit CXCR4+ cells infiltration and neovascularization in periodontal tissue and escape from the immune response.

Estrogen regulates CCR gene expression and function in T lymphocytes.

Estrogen has been implicated in the observed female bias in autoimmune diseases. However, the mechanisms behind this gender dimorphism are poorly defined. We have previously reported that in vivo T cell trafficking is gender- and estrogen-dependent. Chemokine receptors are critical determinants of T cell homing and immune response. In this study, we show that the female gender is associated with increased CD4(+) T cell CCR1-CCR5 gene and protein expression in mice. The increased CCR expression correlates with enhanced in vitro chemotaxis response to MIP-1beta (CCL4). In vivo treatment of young oophorectomized and postmenopausal female mice with 17beta-estradiol also increased CD4(+) T cell CCR expression. Finally, 17beta-estradiol enhances tyrosine phosphorylation in T cells stimulated with MIP-1alpha in a time-dependent manner. Our results indicate an important role of estrogen in determining T cell chemokine response that may help explain the increased susceptibility and severity of autoimmune diseases in females.