Human IP-9: A keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3)

Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-gamma stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-gamma-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-gamma-inducible protein-9, revealed that interferon-gamma-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-gamma-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as beta-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-gamma-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.     

Induction of cytokine (interleukin-1alpha and tumor necrosis factor-alpha) and chemokine (CCL20, CCL27, and CXCL8) alarm signals after allergen and irritant exposure

The immune system is called into action by alarm signals generated from injured tissues. We examined the nature of these alarm signals after exposure of skin residential cells to contact allergens (nickel sulfate and potassium dichromate) and a contact irritant [sodium dodecyl sulfate (SDS)]. Nickel sulfate, potassium dichromate, and SDS were applied topically to the stratum corneum of human skin equivalents. A similar concentration-dependent increase in chemokine (CCL20, CCL27, and CXCL8) secretion was observed for all three chemicals. Exposure to nickel sulfate and SDS was investigated in more detail: similar to chemokine secretion, no difference was observed in the time- and concentration-dependent increase in pro-inflammatory cytokine [interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha)] secretion. Maximal increase in IL-1alpha secretion occurred within 2 h after exposure to both nickel sulfate and SDS and prior to increased chemokine secretion. TNF-alpha secretion was detectable 8 h after chemical exposure. After allergen or irritant exposure, increased CCL20 and CXCL8, but not CCL27, secretion was inhibited by neutralizing human antibodies to either IL-1alpha or TNF-alpha. Our data show that alarm signals consist of primary and secondary signals. IL-1alpha and TNF-alpha are released as primary alarm signals, which trigger the release of secondary chemokine (CCL20 and CXCL8) alarm signals. However, some chemokines, for example, CCL27 can be secreted in an IL-1alpha and TNF-alpha independent manner. Our data suggest that skin residential cells respond to both allergen and irritant exposure by releasing mediators that initiate infiltration of immune responsive cells into the skin.     

Chemokines produced by mesothelial cells: huGRO-α, IP-10, MCP-1 and RANTES

Recently we showed the in vivo relevance of chemokines in cases of bacterial peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients. Mesothelial cells, the most numerous cells in the peritoneal cavity, are hypothesized to function as a main source of chemokine production. We investigated the time- and dose-dependent expression patterns of four chemokines by mesothelial cells at the mRNA and protein level in response to stimulation with physiological doses of proinflammatory mediators that are present at the site of bacterial inflammation. Besides the chemokines huGRO-α (attractant for neutrophils), MCP-1 and RANTES (monocyte attractants), the expression and production of IP-10 was analysed. Mesothelial cells were cultured and stimulated with either IL-1β, tumour necrosis factor-alpha (TNF-α) or IFN-γ or combinations of these. The time- and dose-dependent mRNA expression of the chemokines was determined by Northern blot analysis and the protein production by ELISA. It was concluded that mesothelial cells could indeed be triggered by the mentioned stimuli to induce mRNA and protein production (huGRO-α and IP-10) or to augment constitutive protein production (MCP-1). However, RANTES mRNA and protein production could only be induced in some cases and only in small amounts. The chemokine response of mesothelial cells was regulated differentially, depending on the stimulus and the chemokine measured. In distinct cases, combination of the stimuli led to synergy in mRNA expression and protein production. The presented in vitro data support our hypothesis that mesothelial cells in vivo are the main source of relevant chemokines in response to proinflammatory mediators, suggesting an important role for mesothelial cells in host defence.     

CXCL8 secretion by dendritic cells predicts contact allergens from irritants.

Monocyte-derived dendritic cell functions have been explored for identification of contact allergens in vitro. Current methods, including measurement of changes in cell surface marker expression (e.g. CD83, CD86) do not provide a sensitive method for detecting the sensitising potential of a chemical. In this study, we investigated whether chemokine production by monocyte-derived dendritic cells is increased upon maturation and whether chemokine production can provide methodology for the detection of allergens. Monocyte-derived dendritic cells were exposed to allergens (nickel sulphate, cobalt chloride, palladium chloride, copper sulphate, chrome-(III)-chloride, potassium dichromate, p-phenylenediamine and dinitrochlorobenzene) and irritants (sodium dodecyl sulphate, dimethylsulphoxide, benzalkoniumchloride and propane-1-ol). CD83 and CD86 expression was analysed by flow cytometry and chemokine production (CXCL8, CCL5, CCL17, CCL18, CCL19, CCL20, CCL22) was determined by ELISA. Significant up regulation of CD83 and CD86 expression could only be induced by three out of seven and five out of seven allergens, respectively. In contrast, CXCL8 production was significantly increased after stimulation with all allergens tested, whereas irritant exposure led to decreased CXCL8 production. All other chemokines tested, failed in identifying contact allergens. In conclusion, CXCL8 production, next to CD83 and CD86 up regulation, by monocyte-derived dendritic cells provides a promising in vitro tool for discrimination between allergens and irritants.     

Autocrine regulation of re-epithelialization after wounding by chemokine receptors CCR1, CCR10, CXCR1, CXCR2, and CXCR3

This study identifies chemokine receptors involved in an autocrine regulation of re-epithelialization after skin tissue damage. We determined which receptors, from a panel of 13, are expressed in healthy human epidermis and which monospecific chemokine ligands, secreted by keratinocytes, were able to stimulate migration and proliferation. A reconstructed epidermis cryo(freeze)-wound model was used to assess chemokine secretion after wounding and the effect of pertussis toxin (chemokine receptor blocker) on re-epithelialization and differentiation. Chemokine receptors CCR1, CCR3, CCR4, CCR6, CCR10, CXCR1, CXCR2, CXCR3, and CXCR4 were expressed in epidermis. No expression of CCR2, CCR5, CCR7, and CCR8 was observed by either immunostaining or flow cytometry. Five chemokine receptors (CCR1, CCR10, CXCR1, CXCR2, and CXCR3) were identified, the corresponding monospecific ligands (CCL14, CCL27, CXCL8, CXCL1, CXCL10, respectively) of which were not only able to stimulate keratinocyte migration and/or proliferation but were also secreted by keratinocytes after introducing cryo-wounds into epidermal equivalents. Blocking of receptor-ligand interactions with pertussis toxin delayed re-epithelialization, but did not influence differentiation (as assessed by formation of basal layer, spinous layer, granular layer, and stratum corneum) after cryo-wounding. Taken together, these results confirm that an autocrine positive-feedback loop of epithelialization exists in order to stimulate wound closure after skin injury.