Cell-density-regulated chemotactic responsiveness of keratinocytes in vitro.

Keratinocytes represent the main constituents of the epidermis and have been found to play a regulatory role in a variety of inflammatory skin diseases. The functional activity of keratinocytes is highly heterogeneous, and depends on the cell localization in the epidermal architecture, and the maturation or differentiation state of the cells. Spontaneously proliferating HaCaT cells, showing several similarities to basal epidermal keratinocytes, were found to respond to external chemoattractants, including the chemokines RANTES (regulated on activation normal T cell expressed and secreted) and interleukin-8 and the mu-opioid agonist DAMGO ([d-ala2, N-Me-Phe4, Gly-ol5]enkephalin) in migration assays. The chemotactic responsiveness was highly dependent on the cell density of the monolayer, with greatest chemotactic activity at the highest cell density. Whereas RANTES was found to be the most potent chemoattractant, constitutive RANTES production was also detected in the HaCaT cultures. We found an inverse correlation between constitutive RANTES production and chemotactic responsiveness toward external RANTES, suggesting a possible functional down-modulation of the RANTES receptors, CC chemokine receptor 1 and CC chemokine receptor 5, during culture. Results from confocal laser scanning microscopy showed reduced CC chemokine receptor 1, but not CC chemokine receptor 5, expression by HaCaT cells at low cell densities, which was abolished in the presence of neutralizing antibodies against RANTES. The total CC chemokine receptor 1 pool (surface and intracellular receptors), however, showed no significant change during in vitro culture. Chemotactic responsiveness toward RANTES was directly correlated with the level of CC chemokine receptor 1 surface expression. Taken together these results show that with keratinocyte proliferation and the progressive increase in cell density there are dramatic alterations in keratinocyte function.

The synthetic peptide WKYMVm attenuates the function of the chemokine receptors CCR5 and CXCR4 through activation of formyl peptide receptor-like 1.

The G protein-coupled 7 transmembrane (STM) chemoattractant receptors can be inactivated by heterologous desensitization. Earlier work showed that formly peptide receptor-like 1 (FPRL1), an STM receptor with low affinity for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalamine (fMLF), is activated by peptide domains derived from the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 and its activation results in desensitization and down-regulation of the chemokine receptors CCR5 and CXCR4 from monocyte surfaces. This study investigated the possibility of interfering with the function of CCR5 or CXCR4 as HIV-1 coreceptors by activating FPRL1. Cell lines were established expressing FPRL1 in combination with CD4/CXCR4 or CD4/CCR5 and the effect of a synthetic peptide, WKYMVm, a potent activator of formyl peptide receptors with preference for FPRL1 was determined. Both CXCR4 and CCR5 were desensitized by activation of the cells with WKYMVm via a staurosporine-sensitive pathway. This desensitization of CXCR4 and CCR5 also attenuated their capacity as the fusion cofactors for HIV-1 envelope glycoprotein and resulted in a significant inhibition of p24 production by cell lines infected with HIV-1 that use CCR5 or CXCR4 as coreceptors. Furthermore, WKYMVm inhibited the infection of human peripheral monocyte-derived macrophages and CD4(+) T lymphocytes by R5 or X4 strains of HIV-1, respectively. These results indicate that heterologous desensitization of CCR5 and CXCR4 by an FPRL1 agonist attenuates their major biologic functions and suggest an approach to the development of additional anti-HIV-1 agents. (Blood. 2001;97:2941-2947)

Desensitization of chemokine receptor CCR5 in dendritic cells at the early stage of differentiation by activation of formyl peptide receptors.

Chemokine receptors are subjected to heterologous desensitization by activation of formyl peptide receptors. We investigated the cross-talk between formyl peptide receptors and the chemokine receptor CCR5 in human monocyte-differentiated immature dendritic cells (iDC). Monocytes cultured with GM-CSF and IL-4 for 4 days exhibit markers characteristic of iDC and maintain the expression of both formyl peptide receptors FPR and FPRL1, as well as CCR5. Pretreatment of iDC with W peptide (WKYMVm), a potent agonist for FPR and FPRL1 but with preference for FPRL1, resulted in down-regulation of CCR5 from the cell surface and reduced cell response to the CCR5 ligands through a PKC-dependent pathway. Furthermore, W peptide induced a PKC-dependent phosphorylation of CCR5 and inhibited infection of iDC by R5 HIV-1. Our results indicate that the expression and functions of CCR5 in iDC can be attenuated by W peptide, which activates formyl peptide receptors, and suggest an approach to the design of novel anti-HIV-1 agents.

Mu-opioid induction of monocyte chemoattractant protein-1, RANTES, and IFN-gamma-inducible protein-10 expression in human peripheral blood mononuclear cells.

Strong evidence for the direct modulation of the immune system by opioids is well documented. Mu-opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-gamma-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [D-Ala(2),N:-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO), a mu-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.

Down-regulation of the chemokine receptor CCR5 by activation of chemotactic formyl peptide receptor in human monocytes.

Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)