Astrocytes express functional chemokine receptors.

Multiple lines of evidence are presented characterizing the functional expression of chemokine receptors CXCR4, CCR1, CCR5, and CX3CR1 on astrocytes. Most of these receptors are expressed at low levels and may only be detectable on a subset of cells during disease or following cytokine induction. The expression of CXCR2, CCR2, CCR3, CCR10, CCR11, and several orphan receptors associated with HIV-1 infection has also been proposed. The appearance of several chemokine receptors implies a wider role for chemokines in the regulation of central nervous system functions. Available evidence indicates that selected chemokines induce further chemokine synthesis in astrocytes providing a mechanism to amplify inflammatory responses in the central nervous system.

Modulation of experimental autoimmune encephalomyelitis: effect of altered peptide ligand on chemokine and chemokine receptor expression.

Experimental autoimmune encephalomyelitis (EAE) is a T helper 1 (Th1) cell mediated demyelinating disease and the principal animal model for multiple sclerosis. Spinal cords from SJL mice primed with proteolipid protein peptide 139-151 (pPLP) expressed the chemokines RANTES, MCP-1, MIP-2, KC, MIP-1alpha, MIP-1beta, Mig, and fractalkine. We also identified IP-10 in these samples and described a sequence polymorphism in this transcript. Chemokine expression was specific for tissues of the central nervous system. MCP-1, IP-10, and MIP-2 RNA expression significantly correlated with clinical score. Chemokine receptor expression generally correlated with ligand expression. pPLP-primed mice expressed the Th1-associated markers CCR5 and CXCR3 on mononuclear cells. In addition, cells expressing CCR1, CCR2, CCR3, CCR4, CCR8, and CXCR2 were detected. Here we demonstrate that altered peptide ligand (APL)-induced protection from EAE was accompanied by modulation of chemokine and chemokine receptor expression. Spinal cord tissue sections from APL-protected mice showed greatly reduced levels of all chemokines and of CCR1, CCR5, CCR8, CXCR2 and CXCR3. The Th2-associated chemokine receptors CCR3 and CCR4 were found in protected mice, supporting the hypothesis that Th1 but not Th2 cells are down-regulated by APL treatment. This report concludes that chemokines and chemokine receptors can be useful tools to follow modulation of autoimmune disease.

Cloning and chromosomal mapping of an orphan chemokine receptor: mouse RDC1.

Degenerate RT-PCR was used to identify a new seven-transmembrane-spanning receptor expressed in astrocytes. A receptor, termed RDC1, displaying the characteristic structural features of a chemokine receptor was cloned. The predicted 362-amino-acid sequence displayed 92% and 91% similarity to the human and dog orphan receptor RDC1, respectively. In addition, RDC1 shares 43% amino acid similarity to rabbit and mouse CXCR2. Transcripts of RDC1 were found in astrocytes, heart, kidney, the mesangial tumor line MES-13, spleen, and neutrophils by means of northern blot. Using linkage analysis of interspecies backcross mice, we localized to chromosome 1 the genes for mouse CXCR2, CXCR4, and RDC1. Mouse RDC1 is linked to and lies between the genes for the mouse CXC chemokine receptors CXCR2 and CXCR4. The combined data of chromosomal location and sequence similarity suggest that RDC1 is an orphan CXC chemokine receptor.

Murine astrocytes express a functional chemokine receptor.

Elevated levels of chemokines have been observed in various diseases of the CNS. Little is known, however, about how these chemokines affect parenchymal cells of the CNS. The current studies examine astrocyte chemotaxis to the mouse chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Murine astrocytes demonstrate directed migration along a chemical gradient in response to 10(-10)-10(-8) M MIP-1alpha. Peak chemotactic responses are noted at 10(-9) M. MIP-1alpha-induced astrocyte migration is specifically inhibitable with pertussis toxin, suggesting a role for Galphai proteins in the signaling process. RT-PCR and in situ hybridization were used to identify expression of the murine CCR1 MIP-1alpha receptor on astrocytes. Astrocytes contain mRNA for CCR1, but messages for CCR4 and the orphan chemokine receptor MIP-1alphaR-like#1 were not detected. The combined results suggest that a functional chemokine receptor is expressed on resident cells of the CNS. We speculate that the interactions of chemokines with astrocytes are involved in inflammatory reactions of the CNS.

Functional expression of the CXC-chemokine receptor-4/fusin on mouse microglial cells and astrocytes.

The mRNA for the seven-transmembrane-spanning G protein-coupled receptor fusin/CXCR-4 is expressed in primary mouse astrocyte cultures and the transformed mouse microglial cell line, N9. Cell surface expression of fusin in these cells was confirmed by staining with a polyclonal anti-fusin Ab. The functional capacity of this chemokine receptor was examined by evaluating the calcium responses following stimulation of glial cells with the CXC-chemokine, stromal-derived cell factor-1alpha (SDF-1alpha). Both astrocytes and microglial cells mobilized calcium following stimulation with chemically synthesized SDF-1alpha. SDF-1alpha- and carbachol-mediated calcium responses of astrocytes were partially inhibited by treatment with pertussis toxin (PTx), suggesting receptor coupling to a combination of G alpha(i) and other G proteins. In contrast, the calcium responses of microglial cells to SDF-1alpha were completely PTx sensitive, while responses to carbachol stimulation were PTx resistant. The ability of SDF-1alpha to induce glial cell migration was also examined. Synthetic SDF-1alpha was a potent chemoattractant for mouse microglial cells at ligand concentrations of 10 to 500 ng/ml; peak responses were noted at 100 ng/ml. In contrast, astrocytes did not migrate toward a gradient of SDF-1alpha. The failure of SDF-1alpha to induce astrocyte migration was specific, as another chemokine, macrophage inflammatory protein-1alpha, triggered astrocyte chemotaxis.

Lymphocyte recruitment following spinal cord injury in mice is altered by prior viral exposure.

The inflammatory response induced by mechanical lesion of the spinal cord is known to include the recruitment of neutrophils and macrophages, while the involvement of lymphocytes has been largely ignored. We have studied the pattern of lymphocyte recruitment following partial transection of the mouse spinal cord. Using immunohistochemical techniques, all three types of lymphocytes (CD4-positive T-cells, CD8-positive T-cells and B-cells) were found in the vicinity of the lesion site within hours and persisted for up to 7 days. There was a predominance of B-lymphocytes during the first 3 days. A second, late phase of cell infiltration, dominated by CD8-positive T-lymphocytes, occurred in mice that had been raised in a conventional breeding unit and had acquired antibody titres to a common murine virus (mouse hepatitis virus). In contrast, mice kept in specific pathogen-free facilities did not show this late-phase response. These findings suggest a possible role for lymphocytes in secondary tissue loss, local demyelination, scar formation, cytokine-mediated inflammatory responses or trophic processes. They also provide evidence that a virus infection can significantly enhance the reaction of T-cells to a spinal cord lesion.

Alternate splicing of mouse fusin/CXC chemokine receptor-4: stromal cell-derived factor-1alpha is a ligand for both CXC chemokine receptor-4 isoforms.

Inspection of the intron splice junction of the mouse chemokine receptor fusin/CXC chemokine receptor R-4 (CXCR-4) revealed a potential in-frame alternative splice site in the region encoding the N-terminal ectodomain of the receptor. Both predicted splice products were detected by reverse transcriptase-PCR. Cell lines of T lymphocyte, B lymphocyte, and macrophage lineage, plus populations of elicited peritoneal exudate cells, thymocytes, and astrocytes coexpressed mRNA for both isoforms. The full length cDNA of the shorter alternate splice product, termed CXCR-4B, was cloned from peritoneal exudate cell RNA. Chinese hamster ovary cells transfected with either isoform, CXCR-4A or CXCR-4B, responded to stromal cell-derived factor-1alpha with a rise in intracellular calcium. Both alternate splice products are therefore functional stromal cell-derived factor-1 alpha receptors.

Identification of a new mouse beta-chemokine, thymus-derived chemotactic agent 4, with activity on T lymphocytes and mesangial cells.

Thymus-derived chemotactic agent 4 (TCA4), a new member of the beta-chemokine family, was cloned from a mouse thymic cDNA library. High levels of TCA4 mRNA are expressed in thymus; lower levels of message are found in spleen, heart, and kidney. Anti-TCA4 antibodies were used to localize sites of TCA4 expression within lymphoid tissues. In the thymus, UEA-1+ medullary epithelial cells, some endothelial cells, and additional undefined stromal elements were stained with anti-TCA4. TCA4 was also expressed as a meshlike network in splenic white pulp and in the medullary region of the lymph nodes. In addition, some lymph node and splenic blood vessels stained with anti-TCA4 antibodies. Rel B NFkappaB-deficient mice lack a transcription factor required for the generation of dendritic cells and the development of an organized thymic medulla. Rel B-deficient animals express very low levels of TCA4 in the thymus and little or no TCA4 in the periphery. At subnanomolar concentrations, TCA4 is a chemoattractant of mature T cells; the potential role of this novel chemokine in facilitating normal lymphocyte traffic is discussed. TCA4 is also a chemoattractant of cultured mesangial cells. Neutralizing anti-TCA4 mAb was used to demonstrate the specificity of TCA4-mediated cell migration. Finally, competitive binding studies with a SV40-transformed mouse mesangial cell line demonstrated that other murine beta-chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and thymus-derived chemotactic agent 3) do not compete for TCA4 binding.

Cloning of the mouse fusin gene, homologue to a human HIV-1 co-factor.

Previous studies have demonstrated that mouse cells do not become infected with HIV-1 despite transfection with human CD4. Recently, a human protein termed "fusin" with characteristics of a seven-transmembrane-spanning receptor was found to be a co-factor required for the entry and fusion of HIV-1 with human CD4-bearing lymphocytes. Thus, cloning of the murine homologue of the human fusin (also termed CXCR-4) gene could provide an important comparative tool for identification of the structures crucial for fusin function. Using degenerate PCR, the mouse homologue of human fusin was cloned from a peritoneal exudate cell cDNA library. The predicted amino acid sequence is 91% identical to human fusin. Twenty-eight of the 37 amino acid differences between mouse and human fusin are located in the ectodomains, suggesting that the intracytoplasmic components that mediate G protein binding and signaling are highly conserved. Northern blot analysis showed a message of 2.2 kb in thymus, spleen, neutrophils, and primary astrocyte cultures. Lymphoid and monocyte cell lines also expressed message for fusin. The coding regions of most chemokine receptors lack introns. In contrast, cloning of genomic DNA for mouse fusin revealed the presence of a 2.3-Kb intron separating the first seven amino acids from the remaining 352 residues. Therefore, the mouse fusin gene has a unique genomic organization compared with other chemokine receptors.

Decreased IL-4 production in new onset type I insulin-dependent diabetes mellitus.

IL-4 has been shown to protect against diabetes development in rodent models of insulin-dependent (type I) diabetes mellitus (IDDM). To study IL-4 production in human IDDM, PBMC from IDDM patients and controls were stimulated in vitro with PHA, anti-CD3 mAb, or PMA and ionophore. IL-4 production by PBMC or T cells was strongly impaired in IDDM patients at diabetes onset (p < 0.0001). The mean IL-4 response of patients in the honeymoon stage was higher than the mean of the new onset patients, but significantly lower than the control group (p = 0.01). Patients with IDDM of longer duration (>2 yr) showed a wide range of IL-4 responses and their mean IL-4 response was lower than the controls; however, the difference was not statistically significant. IL-4 mRNA levels were measured using competitive reverse transcription PCR. The results showed greatly reduced mRNA levels in new onset IDDM. In contrast, IL-1 production (measured by ELISA) and IFN-gamma mRNA (measured by reverse transcription PCR) were not significantly different in IDDM. The results suggest an imbalance of inflammatory vs anti-inflammatory cytokine production at the onset of IDDM. Deficient IL-4 production as seen at the onset of IDDM may play a role in the development of diabetes by allowing the inflammatory/autoimmune process in pancreatic islets to progress.

Mouse astrocytes respond to the chemokines MCP-1 and KC, but reverse transcriptase-polymerase chain reaction does not detect mRNA for the KC or new MCP-1 receptor.

Previous studies demonstrated the involvement of astrocytes in the development of astrogliosis, a condition in which these cells undergo proliferation and hypertrophy. To examine whether astrocytes could migrate into lesions, we tested the influence of the murine chemokines MCP-1, KC, TCA3, and MIP-1 beta on migration of cultured neonatal mouse astrocytes. Subnanomolar concentrations of MCP-1 and KC were active chemoattractants indicating that these molecules were effective at physiologic concentrations. Specificity of MCP-1 was demonstrated by antibody inhibition and by the finding that the chemokine MIP-1 beta failed to induce astrocyte migration. The migratory responses were sensitive to pertussis toxin; this finding is consistent with involvement of G protein-coupled receptors. To examine the receptors for these chemokines further, we cloned the mouse homolog of the human MCP-1 receptor from a mouse peritoneal exudate cell cDNA library. The gene had 78% nucleotide sequence homology with the human MCP-1 receptor (the nucleotide sequence of clone 1 encoding the mouse MCP-1 receptor can be obtained from the GenBank database, accession number U56819). However, reverse transcriptase-polymerase chain reaction (RT-PCR) failed to detect message for either the MCP-1 or KC receptors in astrocytes. The combined data suggest that mouse astrocytes use novel receptors to recognize these chemokines.

IL-4 expression in human T cells is selectively inhibited by IL-1 alpha and IL-1 beta.

Imbalances in anti-inflammatory and proinflammatory cytokines may be responsible for initiation or progression of diverse pathologic states including autoimmune and infectious diseases. IL-4 production of proinflammatory cytokines and IL-12 promotes differentiation and activation of IFN-gamma-producing T cells, but does a counter-regulatory effect of proinflammatory cytokines on IL-4 production exist? This study evaluates the effect of proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-12, and TNF-alpha) on IL-4 production in primary human T cell cultures. PBMCs from healthy individuals were tested for IL-4 production in response to PHA and various cytokines. IL-4 was measured by proliferation of the IL-4-sensitive T cell line (CT.h4S) or ELISA. IL-1 alpha and IL-1 beta inhibited IL-4 production by 20 to 80% in > 92% of healthy individuals (p = 0.0001, paired t-test). IL-12 had an inhibitory effect on PBMC IL-4 production as previously described, but neither IL-6 nor TNF-alpha inhibited IL-4 production. IL-1 had no effect on PHA-induced PBMC or purified T cell proliferation or IL-2 production. IL-4 production by purified T cells stimulated by PHA or the combination of PMA with calcium ionophore (A23187) was inhibited by IL-1, and reconstitution with peripheral blood-derived adherent macrophages had no effect. IL-12 did not inhibit IL-4 production in stimulated purified T cells. Steady state IL-4 mRNA levels were determined by semiquantitative competitive reverse transcribed PCR (RT-PCR). Marked inhibition of IL-4 mRNA levels were seen at 5 h after exposure to IL-1. This interaction between IL-1 and IL-4 may be an important physiologic regulator of the balance between proinflammatory cytokines from activated macrophages and anti-inflammatory cytokines from T cells.

HIV type 1 induction of interleukin 1 and 6 production by human thymic cells.

In vitro and in vivo studies have demonstrated that HIV can infect thymocytes at different maturational stages and lead to changes in the thymic microenvironment. To determine the effect of HIV on thymic stromal cells and the production of cytokines important in thymocyte development, three types of adherent thymic cultures were established and studied: thymic epithelial cells (TECs), macrophage-enriched, and mixed cultures of macrophages and TECs (M phi/TEC). Cultures were exposed to HIV-1 strains HIV-1IIIB and HIV-1Ba-L, and studied from day 2 to day 26 for the presence of infection, cytopathology, and cytokine (IL-1 alpha, IL-1 beta, and IL-6) production. M phi/TEC and macrophage-enriched cultures were infected by both HIV strains without cytopathic changes. The TECs grew well in culture for at least 6 weeks and showed no evidence of infection, cytopathology, or changes in cytokine production with HIV. Only cultures containing macrophages (M phi/TEC or macrophage enriched) showed changes in cytokine production with HIV. Sustained production of IL-1 alpha was seen for up to 20 days, with small or no increases in IL-1 beta. M phi/TEC cultures produced high constitutive levels of IL-6 that were not changed by HIV. Unstimulated macrophage-enriched cultures produced small amounts of IL-6 that were increased by HIV 20-fold. This study suggests that HIV infection in vivo can lead to infection of thymic macrophages resulting in cytokine abnormalities and a constant source for HIV to infect maturing thymocytes. These cytokine effects could lead to abnormal maturation and contribute to the lack of regeneration of the mature CD4+ T cell pool.

Lactation from axillary breast tissue in the absence of a supernumerary nipple. A case report.

A case of lactation apparently from axillary supernumerary breast tissue was observed in a lactating woman. The tissue showed histologic features typical of both normal glands of lactation and apocrine sweat glands, and the fluid showed evidence of milk production. The supernumerary breast tissue was not associated with a nipple. Renal and skeletal anomalies were also noted.

HIV-1 infection of macrophages promotes long-term survival and sustained release of interleukins 1 alpha and 6.

HIV infection of macrophages in vivo may result in activation of monokine genes and cause persistent release of immunomodulatory and inflammatory cytokines. Studies that have examined cytokine (IL-1, IL-6, and TNF-alpha) activation by in vitro infection of normal peripheral blood mononuclear cells (PBMCs) with HIV-1 have produced conflicting results. The present study shows that for monokine induction by HIV-1-IIIB preparations derived from the H9 tumor cell line, partial purification of virus particles is essential. Infectious HIV-1 induces the release of high levels of IL-1 alpha, IL-1 beta, and IL-6 bioactivity by adherent PBMCs in the first 3 days following in vitro infection, but only IL-1 alpha and IL-6 continue to be released over several weeks of culture. High levels of bioactive IL-1 beta were released only up to 72 hr following infection, although intracellular IL-1 beta was detectable for at least 3 weeks. No TNF-alpha bioactivity or immunoreactive protein was detectable at > 48 hr in HIV-infected cultures. This time course of monokine release was dependent on the number of infectious particles added to PBMC cultures. In long-term cultures (> 1 month) HIV infection was found to promote the viability of macrophages. The finding of sustained release of IL-1 alpha and IL-6 by infected macrophages, without additional stimulation, suggests that these mediators are released by HIV-1-infected macrophages in AIDS patients, where they may interfere with proper immune regulation.

Mechanism of a lymphocyte abnormality associated with HLA-B8/DR3: role of interleukin-1.

Lymphocytes from normal individuals with the histocompatibility antigens HLA-B8 and DR3 have impaired proliferative responses when stimulated with suboptimal concentrations of mitogens. We have previously shown that an important factor in the impaired response is a failure to produce normal quantities of interleukin-2 (IL-2). To examine the mechanism of decreased responsiveness further, we measured interleukin-1 (IL-1) production of low responder subjects compared with controls. The peripheral blood mononuclear cells of five low responder individuals with HLA-B8/DR3 stimulated with 0.05 micrograms/ml of phytohaemagglutinin (PHA) accumulated only 0.036 U/ml of IL-1 compared with 0.32 U/ml for normal responders. There was a highly significant correlation between the PHA-stimulated IL-1 concentration at 12 h and the subsequent IL-2 concentration at 48 h(r = 0.89, P less than 0.0001) suggesting a role of decreased IL-1 production in the impaired response. A study of unfractionated or column-fractionated culture supernatants revealed no evidence that the decreased IL-1 activity in the supernatants of low responder subjects was related to increased IL-1 inhibitor concentrations. These results suggest that impaired IL-2 production and lymphocyte proliferation in healthy subjects with HLA-B8/DR3 may be mediated at least in part by decreased IL-1 production, and implicates a defect of a very early event in lymphocyte activation.

Interleukin-1 and a 7-kDa T-cell inhibitory monokine reflect disease activity in infection with HIV-1.

Previous studies demonstrated that cultured peripheral blood mononuclear cells (PBMC) from patients with AIDS produce high levels of interleukin 1 (IL-1) and a 7-kDa T-cell inhibitory monokine (TCIM). To determine if the increase in the production of these cytokines corresponded with disease activity, we studied the production of IL-1 and TCIM by PBMC from patients with different stages of human immunodeficiency virus (HIV) infection. Eight patients with asymptomatic seropositive infection, three patients with AIDS-related complex (ARC), three patients with persistent generalized lymphadenopathy (PGL), and six patients meeting the full criteria for diagnosis of AIDS were studied. Patients with AIDS produced increased amounts of TCIM (4.1 times control values, p less than 0.003) and IL-1 (2.0 times control values, p less than 0.05). In contrast, asymptomatic seropositive patients produced less TCIM (0.36 times control values, p less than 0.004) and IL-1 (0.61 times control values, p less than 0.05). Different trends in the levels of these factors produced by patients with ARC and PGL were noted, although results were not statistically significant in general. Patients with ARC tended to produce less IL-1 (0.42 times control values, p less than 0.05), whereas patients with PGL tended to produce increased amounts of IL-1 (1.7 times control values, NS). ARC patients produced a wide range of TCIM values (0.05-2.8 times control values, NS), and patients with PGL tended to produce increased TCIM values, (4.0 times control values, p less than 0.02). No correlations between the levels of IL-1 or TCIM and T-cell subpopulation numbers (CD4 or CD8) or CD4/CD8 ratios were found.(ABSTRACT TRUNCATED AT 250 WORDS)