Expression of mRNA of chemokine receptor CXCR4 in feline mammary adenocarcinoma.

The expression of mRNA of the chemokine receptor CXCR4 in 65 surgically resected mammary adenocarcinomas from cats was investigated by in situ hybridisation. No expression of the receptor's mRNA was detectable in the mammary tissue of healthy cats, but it was expressed in areas adjacent to necrosis, surrounding blood vessels and cells infiltrating the lymphatics of 47 (72.3 per cent) of the 65 samples. There was a significant relationship between lymphatic infiltration by neoplastic cells and the expression of the receptor's mRNA (P < 0.005), but there was no significant relationship between its expression and the one-year survival of the cats.

RANTES-induced chemokine cascade in dendritic cells.

Dendritic cells (DC) are the most potent APCs and the principal activators of naive T cells. We now report that chemokines can serve as activating agents for immature DC. Murine bone marrow-derived DC respond to the CC chemokine RANTES (10-100 ng/ml) by production of proinflammatory mediators. RANTES induces rapid expression of transcripts for the CXC chemokines KC and macrophage inflammatory protein (MIP)-2, the CC chemokines MIP-1beta and MIP-1alpha, and the cytokines TNF-alpha and IL-6. Synthesis of KC, IL-6, and TNF-alpha proteins were also demonstrated. After 4 h, autoinduction of RANTES transcripts was observed. These responses are chemokine specific. Although DC demonstrated weak responses to eotaxin, DC failed to respond to other chemokines including KC, MIP-2, stromal-derived factor-1alpha, MIP-1beta, MIP-1alpha, monocyte chemoattractant protein-1, T cell activation gene 3, or thymus-derived chemotactic agent 4. In addition, RANTES treatment up-regulated expression of an orphan chemokine receptor termed Eo1. Chemokine induction was also observed after treatment of splenic DC and neonatal microglia with RANTES, but not after treatment of thymocytes or splenocytes depleted of adherent cells. TNF-alpha-treated DC lose responsiveness to RANTES. DC from mice deficient for CCR1, CCR3, and CCR5 respond to RANTES, indicating that none of these receptors are exclusively used to initiate the chemokine cascade. RANTES-mediated chemokine amplification in DC may prolong inflammatory responses and shape the microenvironment, potentially enhancing acquired and innate immune responses.

CD40-mediated activation of T cells accelerates, but is not required for, encephalitogenic potential of myelin basic protein-recognizing T cells in a model of progressive experimental autoimmune encephalomyelitis.

CD40 ligand-CD40 interactions are important in the development of experimental autoimmune encephalomyelitis (EAE), but it is unclear whether this interaction is critical for de novo recruitment of T cells, entry of T cells into the central nervous system (CNS), or effector function of T cells in vivo. In this report we define the role of CD40 in a model of progressive EAE that does not depend on epitope spread or recruitment of new myelin-specific T cells into the CNS. Results show that CD40 is not required for trans-migration of activated T cells through the endothelial blood-brain barrier, and in its absence T cells will both enter the CNS and induce disease. However, interaction with CD40 is critical for optimal activation and encephalitogenicity of cloned Th1 cells. In its presence, Th1 cells enter the CNS earlier and induce more severe disease. Inclusion of IL-12 during activation of Th1 cells in the absence of CD40 can override the otherwise suboptimal level of encephalitogenicity observed. The implication of these findings for therapeutic use of agents designed to block this pathway is discussed.

Delayed-type hypersensitivity.

Delayed-type hypersensitivity (DTH) is an in vivo assay of cell-mediated immune function. DTH reactions are often divided into two phases: the sensitization phase, referring to the initial immunization with specific antigen, and the efferent or challenge phase of the DTH response, which usually follows 6 to 14 days after sensitization. This unit details the protocol for stimulating DTH responses in mice to the O-succinimide ester of the hapten 4-hydroxy-3-nitrophenyl acetyl (abbreviated NP-O-Su).

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.

Macrophage inflammatory protein-2 and KC induce chemokine production by mouse astrocytes.

Astrocytes are specialized cells of the CNS that are implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis. In acute and relapsing-remitting experimental allergic encephalomyelitis, the neutrophil chemoattractant CXC chemokines macrophage-inflammatory protein (MIP)-2 and KC are associated with reactive astrocytes in the parenchyma. In vitro treatment of primary astrocyte cultures with nanomolar concentrations of MIP-2 or KC markedly up-regulated expression of the monocyte/T cell chemoattractants monocyte chemoattractant protein-1, inflammatory protein-10, and RANTES by a mechanism that includes stabilization of mRNA. Production of TNF-alpha and IL-6 transcripts were also noted, as was autocrine induction of MIP-2 and KC message. In addition, low levels of MIP-1alpha and MIP-1beta were induced following treatment with MIP-2 or KC. These effects are specific to astrocytes as MIP-2 treatment of microglial cells failed to elicit chemokine production. The astrocyte chemokine receptor for MIP-2 has 2.5 nM affinity for ligand. Astrocytes from CXCR2-deficient mice still respond to KC and MIP-2, indicating the presence of an alternative or novel high affinity receptor for these ligands. We propose that this KC/MIP-2 chemokine cascade may contribute to the persistence of mononuclear cell infiltration in demyelinating autoimmune diseases.

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.

Induction and suppression of an autoimmune disease by oligomerized T cell epitopes: enhanced in vivo potency of encephalitogenic peptides.

T cell epitope peptides derived from proteolipid protein (PLP139-151) or myelin basic protein (MBP86-100) induce experimental autoimmune encephalomyelitis (EAE) in "susceptible" strains of mice (e.g., SJL/J). In this study, we show that the encephalitogenic effect of these epitopes when injected subcutaneously in complete Freund's adjuvant was significantly enhanced if administered to the animal in a multimerized form as a T cell epitope oligomer (i.e., as multiple repeats of the peptide epitope, such as 16-mers). Oligomer-treated SJL/J mice developed EAE faster and showed a more severe progression of the disease than animals treated with peptide alone. In addition, haplotype-matched B10.S mice, "resistant" to EAE induction by peptide, on injection of 16-mers developed a severe form of EAE. Even more striking, however, was the dramatic suppression of incidence and severity of the disease, seen after single intravenous injections of only 50 microg of the PLP139-151 16-mer, administered to SJL/J mice 7 d after the induction of the disease. Although relapse occurred at about day 45, an additional injection several days before that maintained the suppression. Importantly, the specific suppressive effect of oligomer treatment was also evident if EAE was induced with spinal cord homogenate instead of the single peptide antigen. By contrast, the PLP139-151 peptide accelerated rather than retarded the progression of disease.

The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules.

T cell homing to peripheral lymph nodes (PLNs) is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial tethering and rolling via L-selectin, firm adhesion of T cells requires rapid upregulation of lymphocyte function-associated antigen 1 (LFA-1) adhesiveness by a previously unknown pathway that activates a Galpha(i)-linked receptor. Here, we used intravital microscopy of murine PLNs to study the role of thymus-derived chemotactic agent (TCA)-4 (secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) in homing of adoptively transferred T cells from T-GFP mice, a transgenic strain that expresses green fluorescent protein (GFP) selectively in naive T lymphocytes (T(GFP) cells). TCA-4 was constitutively presented on the luminal surface of HEVs, where it was required for LFA-1 activation on rolling T(GFP) cells. Desensitization of the TCA-4 receptor, CC chemokine receptor 7 (CCR7), blocked T(GFP) cell adherence in wild-type HEVs, whereas desensitization to stromal cell-derived factor (SDF)-1alpha (the ligand for CXC chemokine receptor 4 [CXCR4]) did not affect T(GFP) cell behavior. TCA-4 protein was not detected on the luminal surface of PLN HEVs in plt/plt mice, which have a congenital defect in T cell homing to PLNs. Accordingly, T(GFP) cells rolled but did not arrest in plt/plt HEVs. When TCA-4 was injected intracutaneously into plt/plt mice, the chemokine entered afferent lymph vessels and accumulated in draining PLNs. 2 h after intracutaneous injection, luminal presentation of TCA-4 was detectable in a subset of HEVs, and LFA-1-mediated T(GFP) cell adhesion was restored in these vessels. We conclude that TCA-4 is both required and sufficient for LFA-1 activation on rolling T cells in PLN HEVs. This study also highlights a hitherto undocumented role for chemokines contained in afferent lymph, which may modulate leukocyte recruitment in draining PLNs.

The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer's patch high endothelial venules.

Chemokines have been hypothesized to contribute to the selectivity of lymphocyte trafficking not only as chemoattractants, but also by triggering integrin-dependent sticking (arrest) of circulating lymphocytes at venular sites of extravasation. We show that T cells roll on most Peyer's patch high endothelial venules (PP-HEVs), but preferentially arrest in segments displaying high levels of luminal secondary lymphoid tissue chemokine (SLC) (6Ckine, Exodus-2, thymus-derived chemotactic agent 4 [TCA-4]). This arrest is selectively inhibited by functional deletion (desensitization) of CC chemokine receptor 7 (CCR7), the receptor for SLC and for macrophage inflammatory protein (MIP)-3beta (EBV-induced molecule 1 ligand chemokine [ELC]), and does not occur in mutant DDD/1 mice that are deficient in these CCR7 ligands. In contrast, pertussis toxin-sensitive B cell sticking does not require SLC or MIP-3beta signaling, and occurs efficiently in SLC(low/-) HEV segments in wild-type mice, and in the SLC-negative HEVs of DDD/1 mice. Remarkably, sites of T and B cell firm adhesion are segregated in PPs, with HEVs supporting B cell accumulation concentrated in or near follicles, the target domain of most B cells entering PPs, whereas T cells preferentially accumulate in interfollicular HEVs. Our findings reveal a fundamental difference in signaling requirements for PP-HEV recognition by T and B cells, and describe an unexpected level of specialization of HEVs that may allow differential, segmental control of lymphocyte subset recruitment into functionally distinct lymphoid microenvironments in vivo.

Cutting edge: ectopic expression of the chemokine TCA4/SLC is sufficient to trigger lymphoid neogenesis.

To test whether accumulation of naive lymphocytes is sufficient to trigger lymphoid development, we generated mice with islet expression of the chemokine TCA4/SLC. This chemokine is specific for naive lymphocytes and mature dendritic cells (DC) which express the CCR7 receptor. Islets initially developed accumulations of T cells with DC, with scattered B cells at the perimeter. These infiltrates consolidated into organized lymphoid tissue, with high endothelial venules and stromal reticulum. Infiltrate lymphocytes showed a naive CD44low CD25- CD69- phenotype, though half were CD62L negative. When backcrossed to RAG-1 knockout, DC were not recruited. Interestingly, islet lymphoid tissue developed in backcrosses to Ikaros knockout mice despite the absence of normal peripheral nodes. Our results indicate that TCA4/SLC can induce the development and organization of lymphoid tissue through diffential recruitment of T and B lymphocytes and secondary effects on stromal cell development.

Abundant empty class II MHC molecules on the surface of immature dendritic cells.

A monoclonal antibody specific for the empty conformation of class II MHC molecules revealed the presence of abundant empty molecules on the surface of spleen- and bone marrow-derived dendritic cells (DC) among various types of antigen-presenting cells. The empty class II MHC molecules are developmentally regulated and expressed predominantly on immature DC. They can capture peptide antigens directly from the extracellular medium and present bound peptides to antigen-specific T lymphocytes. The ability of the empty cell-surface class II MHC proteins to bind peptides and present them to T cells without intracellular processing can serve to extend the spectrum of antigens able to be presented by DC, consistent with their role as sentinels in the immune system.

Chemokine amplification in mesangial cells.

Mesangial cells are specialized cells of the renal glomerulus that share some properties of vascular smooth muscle cells and macrophages. They are implicated in the pathogenesis of many forms of nephritis. The murine CXC-chemokines macrophage inflammatory protein-2 (MIP-2) and KC induce migration of mouse mesangial cells. Mesangial cells also exhibit a unique chemokine feedback mechanism. Treatment with nanomolar concentrations of MIP-2 or KC markedly up-regulates monocyte chemoattractant protein-1 and RANTES expression in mesangial cells. Autoinduction of MIP-2 and KC mRNA was also noted. Low levels of MIP-1alpha, MIP-1beta, and IFN-gamma-inducible protein-10 were induced following treatment with higher doses of MIP-2 or KC. These effects are specific to mesangial cells, as MIP-2 or KC treatment of renal cortical epithelial cells or peritoneal macrophages failed to induce chemokine production. This cascade of chemokine interactions may contribute to renal infiltration and leukocyte activation. The abilities of MIP-2 or KC to stimulate their own synthesis may also contribute to the maintenance and chronic course of glomerular inflammation. The mesangial cell receptor for MIP-2 and/or KC is unknown but is not CXC-chemokine receptor-2.

Antigen presenting capacity of brain microvasculature in altered peptide ligand modulation of experimental allergic encephalomyelitis.

Co-immunization with an altered peptide ligand (LR) partially protects SJL mice from proteolipid protein peptide 139-151-induced experimental allergic encephalomyelitis [Kuchroo, V.K., Greer, J.M., Kaul, D., Ishioka, G.Y., Franco, A., Sette, A., Sobel, R.A., Lees, M.B., 1994. A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediated by a diverse T cell repertoire. J. Immunol. 153, 3326-3336; Santambrogio, L., Lees, M.B., Sobel, R.A., 1998. Altered peptide ligand modulation of experimental allergic encephalomyelitis: immune responses within the CNS. J. Neuroimmunol. 81, 1-13]. Clinical protection was noted despite extensive central nervous system inflammation observed after co-immunization with native and altered peptides. To extend our previous reports on this model, we now compare MHC class II expression and antigen presenting cell activity of cells associated with the blood-brain barrier in diseased and protected mice. Immunohistochemical studies identified MHC class II products on both the endothelial and microglial/macrophage populations. Ex vivo experiments suggested a correlation between the reduced clinical disease observed in the co-immunized mice and the antigen presenting activity of cells at the blood-brain barrier. The results suggest that antigen presenting activity is primarily mediated by macrophage-lineage cells of the central nervous system.

Differential recognition of MBP epitopes in BALB/c mice determines the site of inflammatory disease induction.

Although myelin basic protein (MBP)-recognizing T cells are not readily obtained after immunization of BALB/c mice with MBP (reflecting the BALB/c resistance to actively induced experimental autoimmune encephalomyelitis (EAE)), they can be expanded and cloned after several rounds of in vitro culture. The majority of BALB/c-derived clones recognize an epitope defined by MBP peptide 59-76. When transferred to naive BALB/c recipients, these clones cause classical EAE, with characteristic inflammation and demyelination of the central nervous system (CNS). We previously showed that two related clones recognizing a minor epitope, defined by MBP peptide 151-168, cause inflammation and demyelination preferentially of the peripheral nervous system (PNS). Because MBP has alternatively spliced isoforms, residues 151-168 are not present contiguously in all MBP isoforms. In order to determine whether induction of PNS disease is idiosyncratic to these sister clones, or related to their properties of epitope recognition, an independent T-cell line with similar recognition properties was studied. Clone 116F, derived from a BALB/c shiverer mouse, expresses a different T-cell receptor (TCR), with distinct TCR contact residues, but like the previously described T cells, this clone requires residues from both exons 6 and 7 for optimal stimulation. When adoptively transferred to BALB/c recipients, this clone preferentially induces disease of the PNS. A control BALB/c shiverer-derived MBP 59-76-recognizing clone, in contrast, induces CNS disease. These data strongly suggest that the site of disease initiation may correlate with epitope recognition, particularly when alternative isoforms are involved.

The orphan seven-transmembrane receptor apj supports the entry of primary T-cell-line-tropic and dualtropic human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) enters target cells by sequential binding to CD4 and specific seven-transmembrane-segment (7TMS) coreceptors. Viruses use the chemokine receptor CCR5 as a coreceptor in the early, asymptomatic stages of HIV-1 infection but can adapt to the use of other receptors such as CXCR4 and CCR3 as the infection proceeds. Here we identify one such coreceptor, Apj, which supported the efficient entry of several primary T-cell-line tropic (T-tropic) and dualtropic HIV-1 isolates and the simian immunodeficiency virus SIVmac316. Another 7TMS protein, CCR9, supported the less efficient entry of one primary T-tropic isolate. mRNAs for both receptors were present in phytohemagglutinin- and interleukin-2-activated peripheral blood mononuclear cells. Apj and CCR9 share with other coreceptors for HIV-1 and SIV an N-terminal region rich in aromatic and acidic residues. These results highlight properties common to 7TMS proteins that can function as HIV-1 coreceptors, and they may contribute to an understanding of viral evolution in infected individuals.

T-cell responses to myelin basic protein in normal and MBP-deficient mice.

BALB/c mice are resistant to the development of experimental autoimmune encephalomyelitis (EAE) after immunization with myelin basic protein (MBP). Previous studies of BALB/c mice suggest that MBP-specific T-cells can eventually be cloned from these mice, although they are either initially present in very low frequencies or are functionally anergic. To determine what role endogenous MBP expression plays in shaping the BALB/c T-cell repertoire, MBP-deficient BALB/c mice were constructed by breeding the shiverer (shi/shi) mutation onto the BALB/c background. These mice lack all conventional isoforms of MBP due to a deletion of MBP exons 3-7. Studies of the MBP-directed response of these mice suggest that endogenous MBP expression is directly responsible for EAE resistance in BALB/c mice, by quantitatively affecting expression of the T-cell repertoire. In contrast to wild-type BALB/c T-cells, uncloned T-cells from BALB/c shi/shi mice immunized with MBP proliferate in vitro to MBP and MBP peptides 59-76 and 89-101 and are able to induce severe EAE upon transfer to BALB/c recipients expressing MBP.

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.

Use of murine CXCR-4 as a second receptor by some T-cell-tropic human immunodeficiency viruses.

The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.

The CXC-chemokine, H174: expression in the central nervous system.

H174 is a new member of the CXC-chemokine family. A cDNA probe containing the entire H174 coding region recognized a predominant inducible transcript of approximately 1.5 kb expressed in interferon (IFN) activated astrocytoma and monocytic cell lines. H174 message can be induced following IFN-alpha, IFN-beta, or IFN-gamma stimulation. H174 message was also detected in IFN treated cultures of primary human astrocytes, but was absent in unstimulated astrocytes. H174, like IP10 and Mig, lacks the ELR sequence associated with the neutrophil specificity characteristic of most CXC-chemokines. Preliminary experiments suggest H174, IP10 and Mig are independently regulated. Recombinant H174 is a weak chemoattractant for monocyte-like cells. H174 can also stimulate calcium flux responses. The data support the classification of H174 as a member of a subfamily of interferon-gamma inducible non-ELR CXC-chemokines. Brain tissues were obtained at autopsy from one patient with AIDS dementia, one patient with multiple sclerosis, and two normal control patients. H174 and Mig were detected by RT-PCR in brain tissue cDNA derived from the patients with pathological conditions associated with activated astrocytes but not in cDNA from control specimens.