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1.
Scand J Immunol ; 65(5): 479-86, 2007 May.
Article in English | MEDLINE | ID: mdl-17444959

ABSTRACT

CD40-mediated interactions play an important role in the response to a variety of diseases, including cancer. Engagement of CD40 on antigen-presenting cells, namely dendritic cells (DC), by CD40L leads to maturation and up-regulation of co-stimulatory molecules B7.1 and B7.2 (CD80 and CD86). These molecules are requisite to subsequent antigen-specific activation of T cells. T-cell activation is a critical aspect of specific anti-tumour immune responses that have become the focus of a variety of cancer immunotherapy approaches. Clinical trials involving immunologic interventions have shown clinical responses confirming that the immune system can be harnessed for the treatment of cancer. However, the clinical response rate has been low, signifying the need for new immunotherapeutic strategies. To this end, an agonist antibody specific for CD40, CP-870,893, has been developed. A fully autologous mixed tumour cell/lymph node cell model was utilized to demonstrate that CP-870,893 promotes the responsiveness of lymph node-derived T cells to autologous tumour. Specifically, T cells from the tumour-draining lymph nodes are not responsive to autologous tumour cells; however, in the presence of CP-870,893, this unresponsiveness is reversed, as indicated by lymph node cell proliferation and cytokine secretion. Monocyte-derived DC treated with CP-870,893 consistently display a mature phenotype: up-regulation of CD80, CD83, CD86 and HLA-DR expression, increased Mip1alpha and IL-12 secretion, and the loss of exogenous antigen-presenting capability subsequent to treatment with the antibody. These data indicate that CP-870,893 binds to and activates DC, ultimately driving a specific anti-tumour T-cell response.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibody Specificity , Antineoplastic Agents/therapeutic use , CD40 Antigens/immunology , Cell Differentiation/immunology , Dendritic Cells/immunology , Lymph Nodes/immunology , T-Lymphocyte Subsets/immunology , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal, Humanized , CD40 Antigens/agonists , Cell Proliferation , Cells, Cultured , Dendritic Cells/cytology , Epitopes, T-Lymphocyte/immunology , Humans , Lymph Nodes/cytology , Lymphocyte Culture Test, Mixed , T-Lymphocyte Subsets/pathology
2.
Cytokine ; 18(4): 184-90, 2002 May 21.
Article in English | MEDLINE | ID: mdl-12126640

ABSTRACT

CCR2, and its principle ligand MCP-1/CCL2, have been well documented for their ability to induce monocyte infiltration and promote the pathogenesis of rheumatoid arthritis and atherosclerosis. In order to assess additional roles for CCR2, we inserted allogeneic implants into CCR2-/- and MCP-1-/- mice and characterized T cell responses and the regulatory role of CCR2 on MCP-1 expression. The results demonstrate a marked decrease in lymphocyte infiltration in both CCR2-/- and MCP-1-/- animals. In contrast, IL-12 and CTL function were only suppressed in CCR2-/- animals. Further, whereas MCP-1 was only transiently elevated in the inflammatory fluid of WT animals, levels were sustained within the implants (5000pg/ml; >8 days) and serum (243pg/ml) of CCR2-/- mice. Higher levels of MCP-1 were also observed in the culture supernatants of CCR2-/- macrophages as compared to WT cells despite no difference in mRNA levels. Evidence that MCP-1 levels are regulated by receptor binding and internalization was suggested by its rapid decline when added to WT macrophages at 37 degrees C but not 4 degrees C. These studies indicate that CCR2 plays an important role in regulating T cell responses and controlling the level of MCP-1 at inflammatory sites.


Subject(s)
Chemokine CCL2/biosynthesis , Isoantigens , Receptors, Chemokine/biosynthesis , Receptors, Chemokine/physiology , T-Lymphocytes/cytology , Animals , Genotype , Inflammation , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Interleukin-12/metabolism , Interleukin-4/biosynthesis , Lymphocyte Activation , Lymphocytes/metabolism , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neutrophils/metabolism , Phenotype , Receptors, CCR2 , Ribonucleases/metabolism , Temperature , Time Factors
3.
Cancer Immunol Immunother ; 50(3): 125-33, 2001 May.
Article in English | MEDLINE | ID: mdl-11419179

ABSTRACT

The T-cell-specific receptor, CTLA-4, has been demonstrated to be a potent negative regulator of lymphocyte activation, the functional significance of which has been demonstrated in murine tumor models using blocking antibodies. However, the mechanism(s) involved in enhancing tumor regression has not been identified. In this study, we determined whether IFN gamma was playing a role in this activity. In vitro, anti-CTLA-4 enhanced IFN gamma production by lymph node cells obtained from tumor-bearing mice (351 pg/ml vs 77 pg/ml). Additionally, fibrosarcoma-challenged animals treated with anti-CTLA-4 had elevated levels of the IFN-inducible enzyme 2-5-OAS in draining lymph nodes (850 pM vs 260 pM for controls) and an increased amount of IFN gamma in tumor lysates (at day 7, 620 pg/100 micrograms vs 160 pg/100 micrograms in controls). The importance of IFN gamma was demonstrated by the ability of neutralizing antibodies to completely abrogate the anti-tumor effects of anti-CTLA-4. Moreover, fibrosarcoma cells were shown to be exquisitely sensitive to IFN gamma-mediated class I upregulation and histological examination of tumors from anti-CTLA-4-treated mice revealed a trend toward increased tumor cell apoptosis and decreased angiogenesis. These studies have demonstrated that one mechanism for the anti-tumor effects of anti-CTLA-4 relates to its ability to augment IFN gamma production, resulting in an increased expression of class I on the tumor, enhanced apoptosis, and a decrease in blood vessel growth.


Subject(s)
Antigens, Differentiation/metabolism , Immunoconjugates , Interferon-gamma/metabolism , 2',5'-Oligoadenylate Synthetase/metabolism , Abatacept , Animals , Antigens, CD , Antigens, Differentiation/blood , Antigens, Differentiation/immunology , Apoptosis , CTLA-4 Antigen , Cancer Vaccines , Dose-Response Relationship, Drug , Female , Fibrosarcoma/therapy , Flow Cytometry , Immunoglobulin G/metabolism , Immunosuppressive Agents/pharmacology , Immunotherapy , Interferon-gamma/biosynthesis , Lymph Nodes/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mitosis , Neoplasm Transplantation , Neoplasms, Experimental/prevention & control , Neoplasms, Experimental/therapy , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , Time Factors , Up-Regulation
4.
Genomics ; 73(1): 28-37, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11352563

ABSTRACT

We describe the genomic organization of a recently identified CC chemokine, MIP3alpha/CCL20 (HGMW-approved symbol SCYA20). The MIP-3alpha/CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISH analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3alpha/CCL20, Ala MIP-3alpha/CCL20 and Ser MIP-3alpha/CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ala MIP-3alpha/CCL20 or Ser MIP-3alpha/CCL20. Both forms of MIP-3alpha/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-2-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3alpha/CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3alpha/CCL20 and Ala MIP-3alpha/CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known.


Subject(s)
Alternative Splicing/genetics , Chemokines, CC/genetics , Chromosomes, Human, Pair 2 , Macrophage Inflammatory Proteins/genetics , Receptors, Chemokine , Base Sequence , Calcium/metabolism , Chemokine CCL20 , Chemokines, CC/chemical synthesis , Chemokines, CC/physiology , Chromosome Mapping , Cloning, Molecular , DNA , Exons , Humans , In Situ Hybridization, Fluorescence/methods , Introns , Macrophage Inflammatory Proteins/chemical synthesis , Macrophage Inflammatory Proteins/physiology , Molecular Sequence Data , Receptors, CCR6 , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
5.
Transplantation ; 72(12): 1957-67, 2001 Dec 27.
Article in English | MEDLINE | ID: mdl-11773896

ABSTRACT

BACKGROUND: Graft rejection after liver transplantation is associated with a lymphocytic infiltrate, the nature of which will be determined by, among various factors, the local activity of chemokines that attract particular subsets of effector cells to the graft. METHODS: The expression of chemokines and receptors in human liver allografts was studied by immunohistochemistry of tissue and flow cytometry of blood and liver-derived lymphocytes. Receptor function was assessed with in vitro chemotaxis. RESULTS: We report increased expression of chemokine receptors CXCR3, CXCR4, and CCR5 on circulating and graft-infiltrating lymphocytes after liver transplantation. Liver-derived T cells responded to the ligands for these receptors in vitro, which suggests that the receptors are functionally active. The chemokine ligands for these receptors were detected in rejecting allografts. CXCR3 ligands interferon-inducible protein 10 and monokine-induced by gamma interferon were detected on sinusoidal endothelium and interferon-inducible T-cell alpha chemoattractant was detected on portal and hepatic vascular endothelium, whereas the CXCR4 ligand, stromal-derived factor (SDF), was restricted to biliary epithelium. CCR5 ligands have previously been shown on portal endothelium. An in vitro model of T-cell alloactivation demonstrated a similar pattern of expression of functional CXCR3, CXCR4, and CCR5 on T cells. Increased expression of chemokine receptors, especially CCR3 and CCR5, was associated with redistribution of activated Kupffer cells in rejecting grafts. CONCLUSIONS: The patterns of chemokine expression in liver allografts during rejection suggest that the recruitment and positioning of lymphocytes is mediated by specific chemokines. Although ligands for the receptors CXCR3 and CCR5 are important for recruitment, the restriction of SDF to bile ducts suggests that CXCR4 may be involved in the retention of alloactivated lymphocytes at sites of graft damage.


Subject(s)
Chemokines/metabolism , Graft Rejection/complications , Hepatitis/etiology , Hepatitis/metabolism , Liver Transplantation , Receptors, Chemokine/metabolism , Chemokine CXCL10/metabolism , Chemokine CXCL12 , Chemokines, CXC/metabolism , Graft Rejection/metabolism , Graft Rejection/pathology , Humans , Interferon-alpha/metabolism , Kupffer Cells/metabolism , Liver/pathology , Lymphocytes/metabolism , Lymphocytes/pathology , Postoperative Period , Receptors, CXCR3 , Receptors, CXCR4/metabolism , Receptors, CXCR5 , Receptors, Cytokine/metabolism , T-Lymphocytes/metabolism
6.
J Immunol ; 164(10): 5207-14, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10799880

ABSTRACT

Recent studies have suggested a pivotal role for secondary lymphoid chemokine (SLC) in directing dendritic cell trafficking from peripheral to lymphoid tissues. As an extension of these studies, we examined the consequences of anti-SLC Ab treatment during Ag priming on T cell function in an inflammatory response. We used a model of T cell-mediated inflammation, contact hypersensitivity (CHS), where priming of the effector T cells is dependent upon epidermal dendritic cell, Langerhans cells, and migration from the hapten sensitization site in the skin to draining lymph nodes. A single injection of anti-SLC Ab given at the time of sensitization with FITC inhibited Langerhans cell migration into draining lymph nodes for at least 3 days. The CHS response to hapten challenge was inhibited by anti-SLC Ab treatment in a dose-dependent manner. Despite the inhibition of CHS, T cells producing IFN-gamma following in vitro stimulation with anti-CD3 mAb or with hapten-labeled cells were present in the skin-draining lymph nodes of mice treated with anti-SLC Ab during hapten sensitization. These T cells were unable, however, to passively transfer CHS to naive recipients. Animals treated with anti-SLC Ab during hapten sensitization were not tolerant to subsequent sensitization and challenge with the hapten. In addition, anti-SLC Ab did not inhibit CHS responses when given at the time of hapten challenge. These results indicate an important role for SLC during sensitization for CHS and suggest a strategy to circumvent functional T cell priming for inflammatory responses through administration of an Ab inhibiting dendritic cell trafficking.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Chemokines, CC/immunology , Dermatitis, Contact/immunology , Haptens/immunology , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Cell Migration Inhibition , Chemokine CCL21 , Cytokines/biosynthesis , Dermatitis, Contact/etiology , Dermatitis, Contact/prevention & control , Dinitrofluorobenzene/administration & dosage , Dinitrofluorobenzene/immunology , Female , Haptens/administration & dosage , Immune Tolerance/immunology , Immunization , Injections, Intravenous , Langerhans Cells/cytology , Langerhans Cells/immunology , Mice , Mice, Inbred BALB C , Oxazolone/administration & dosage , Oxazolone/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/transplantation
7.
J Immunol ; 164(6): 3392-401, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10706735

ABSTRACT

Macrophage inflammatory protein (MIP-1 alpha), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1 alpha have been well documented, the in vivo biological activities of MIP-1 alpha in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1 alpha and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10-24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1 alpha stimulation. These studies substantiate the biological effects of MIP-1 alpha on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1 alpha to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.


Subject(s)
Cell Movement/immunology , Macrophage Inflammatory Proteins/administration & dosage , Monocytes/immunology , Neutrophils/immunology , Adolescent , Adult , Cell Line , Chemokine CCL4 , E-Selectin/biosynthesis , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Female , Humans , Injections, Intradermal , Macrophage Inflammatory Proteins/pharmacology , Macrophage Inflammatory Proteins/physiology , Male , Middle Aged , Neutrophils/metabolism , Receptors, CCR1 , Receptors, CCR5/biosynthesis , Receptors, Chemokine/biosynthesis , Skin/cytology
8.
Inflammation ; 23(1): 75-86, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10065763

ABSTRACT

The in vitro chemotactic activity of chemokines have been well documented. However, study of their in vivo effects where components of rolling, adherence and diapedesis are pre-requisites to leukocyte infiltration have not been examined in higher species. In this study, we examined the biological activity of the CC chemokine, MIP-1alpha, in rhesus monkeys. Following an intradermal injection, a significant cellular infiltrate and an increase in the number of inflamed vessels were observed. This response peaked at 24 h and was sustained for up to 48 hours after injection. Phenotypically, the specific infiltrate consisted exclusively of CD68+ monocytes with no increase in other cell types over the saline injected controls. These studies represent the first examination of the in vivo effects of MIP-1alpha in higher species and indicate that MIP-1alpha is a selective monocyte recruiting agent in vivo.


Subject(s)
Chemotaxis, Leukocyte , Macaca mulatta/physiology , Macrophage Inflammatory Proteins/pharmacology , Monocytes/drug effects , Monocytes/physiology , Animals , Chemokine CCL3 , Chemokine CCL4 , Chemotaxis, Leukocyte/physiology , Humans , Immunohistochemistry , Injections, Intradermal
9.
Lab Invest ; 78(10): 1239-44, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9800949

ABSTRACT

Experimental allergic encephalomyelitis is a murine model of preclinical autoimmune disease that has pathologic similarities to multiple sclerosis (MS). Although CD4+ T cells have been shown to play a crucial role in the development of disease, we recently demonstrated a link between the development of paralysis and eosinophil infiltration into the spinal cord. As such, CD4+ cells may initiate disease, but eosinophils may be the actual effector cells responsible for causing damage to myelin and causing paralysis. Because MS patients sometimes experience early visual problems, ie, optic neuritis, we explored whether an early eosinophil infiltrate was also observed in the optic nerves of SJL mice after the passive transfer of encephalitogenic T cells. Seven days after the passive transfer of myelin basic protein (MBP)-reactive T cell blasts, we observed a significant infiltration of eosinophils into the optic nerves of the mice. This infiltration persisted during the early phases of paralysis, then declined to baseline values by the peak of limb paralysis on Day 10, and remained at baseline during the remission phase of the disease. Remyelination of optic nerves was observed at this time. These results suggest that eosinophil infiltration into the optic nerve is one of the earliest events occurring after the passive transfer of encephalitogenic T cells in murine experimental allergic encephalomyelitis.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/pathology , Neutrophils/physiology , Optic Nerve/pathology , Animals , CD4-Positive T-Lymphocytes/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Female , Immunization, Passive , Lymphocyte Transfusion , Mice , Mice, Inbred Strains , Myelin Basic Protein/immunology , Neutrophils/pathology , Optic Nerve/physiopathology , Optic Nerve/ultrastructure , T-Lymphocytes , Time Factors
10.
J Exp Med ; 188(3): 603-8, 1998 Aug 03.
Article in English | MEDLINE | ID: mdl-9687537

ABSTRACT

Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1alpha. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes. In this paper, we have investigated the chemokine receptor usage and downstream signaling pathways of HCC-1. Cross-desensitization experiments using THP-1 cells suggested that HCC-1 and MIP-1alpha activated the same receptor. Experiments using a panel of cloned chemokine receptors revealed that HCC-1 specifically activated C-C chemokine receptor (CCR)1, but not closely related receptors, including CCR5. HCC-1 competed with MIP-1alpha for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1alpha). Similarly, HCC-1 was less potent than MIP-1alpha in inducing inhibition of adenylyl cyclase in CCR1-transfected cells. HCC-1 induced chemotaxis of freshly isolated human monocytes, THP-1 cells, and CCR1-transfected cells, and the optimal concentration for cell migration (100 nM) was approximately 100-fold lower than that of MIP-1alpha (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes.


Subject(s)
Chemokines, CC/metabolism , Monocytes/metabolism , Receptors, Chemokine/metabolism , Cell Line , Chemokine CCL3 , Chemokine CCL4 , Chemokines, CC/chemical synthesis , Chemotaxis , Humans , Macrophage Inflammatory Proteins/metabolism , Monocytes/physiology , Receptors, CCR1 , Receptors, Chemokine/genetics , Second Messenger Systems
11.
J Immunol ; 161(3): 1204-11, 1998 Aug 01.
Article in English | MEDLINE | ID: mdl-9686580

ABSTRACT

Superantigens such as staphylococcal enterotoxin A and B (SEA and SEB) activate the immune system by stimulating a large proportion of T lymphocytes through specific Vbeta regions of the TCR and activating macrophages by binding to MHC class II molecules. While the mechanisms by which superantigens activate T lymphocytes have been elucidated, their role in the generation of local immune responses to bacterial invasion is still unclear. In this study we have examined the ability of the superantigens SEA and SEB to elicit an inflammatory reaction in vivo, in s.c. air pouches in the mouse. Upon injection into the s.c. air pouch, the two superantigens stimulated a time-dependent increase in the number of leukocytes appearing in the pouch exudate. The leukocytes migrating into the pouch exudate were predominantly neutrophils, with some mononuclear phagocytes and eosinophils present. No T lymphocytes were detected either in the pouch lining tissue or in the exudate cells. Injection of SEA resulted in increased ICAM-1 expression, as detected by immunohistochemistry, on endothelial cells in the tissue surrounding the air pouch and accumulation of TNF-alpha and the chemokines macrophage inflammatory protein-2 (MIP-2), MIP-1alpha, and JE in the pouch exudate. In addition, pretreatment of mice with Abs raised against ICAM-1, TNF-alpha, MIP-2, MIP-1alpha, KC, or JE inhibited leukocyte accumulation induced by SEA. These data demonstrate that bacterial superantigens may promote inflammation at extravascular sites in vivo, and that this response is secondary to the generation of inflammatory mediators, including chemokines.


Subject(s)
Chemokines/physiology , Intercellular Adhesion Molecule-1/physiology , Staphylococcal Infections/immunology , Staphylococcal Infections/pathology , Staphylococcus aureus/immunology , Superantigens/administration & dosage , Tumor Necrosis Factor-alpha/physiology , Acute Disease , Animals , Cell Movement/immunology , Chemokines/biosynthesis , Enterotoxins/administration & dosage , Inflammation/immunology , Injections, Subcutaneous , Intercellular Adhesion Molecule-1/biosynthesis , Leukocytes/immunology , Male , Mice , Mice, Inbred BALB C , Superantigens/immunology , Tumor Necrosis Factor-alpha/biosynthesis
12.
J Exp Med ; 187(12): 2009-21, 1998 Jun 15.
Article in English | MEDLINE | ID: mdl-9625760

ABSTRACT

Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2-activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes. I-TAC interacts selectively with CXCR3, which is the receptor for two other IFN-inducible chemokines, the IFN-gamma-inducible 10-kD protein (IP-10) and IFN-gamma- induced human monokine (HuMig), but with a significantly higher affinity. In addition, higher potency and efficacy of I-TAC over IP-10 and HuMig is demonstrated by transient mobilization of intracellular calcium as well as chemotactic migration in both activated T cells and transfected cell lines expressing CXCR3. Stimulation of astrocytes with IFN-gamma and IL-1 together results in an approximately 400,000-fold increase in I-TAC mRNA expression, whereas stimulating monocytes with either of the cytokines alone or in combination results in only a 100-fold increase in the level of I-TAC transcript. Moderate expression is also observed in pancreas, lung, thymus, and spleen. The high level of expression in IFN- and IL-1-stimulated astrocytes suggests that I-TAC could be a major chemoattractant for effector T cells involved in the pathophysiology of neuroinflammatory disorders, although I-TAC may also play a role in the migration of activated T cells during IFN-dominated immune responses.


Subject(s)
Chemokines, CXC/metabolism , Lymphocyte Activation , Receptors, Chemokine/metabolism , T-Lymphocytes/immunology , Amino Acid Sequence , Astrocytes , Base Sequence , Calcium/metabolism , Chemokine CXCL11 , Chemokines, CXC/genetics , Chemotaxis, Leukocyte , Chromosomes, Human, Pair 4 , Cloning, Molecular , DNA, Complementary/genetics , Desensitization, Immunologic , Humans , Interferon-gamma/pharmacology , Molecular Sequence Data , Protein Binding , RNA, Messenger/biosynthesis , Receptors, CXCR3 , Sequence Analysis, DNA , Sequence Homology, Amino Acid , T-Lymphocytes/drug effects
13.
Clin Exp Immunol ; 110(3): 397-402, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9409642

ABSTRACT

Antigen stimulation of T cells results in a series of biochemical events including the interaction of both SH2 domains of ZAP-70 with phosphorylated ITAMS on the T cell receptor. In order to study the physiological relevance of decreasing native ZAP-70-SH2 interaction in vivo, we generated transgenic mice expressing a T cell-specific, dominant negative form of ZAP-70 consisting of only the tandem SH2 domains (ZAP-NC). Phenotypically, these animals had a comparable distribution of lymphocyte subsets in the thymus and spleen compared with the wild-type (WT) controls. However, examination of peripheral blood revealed a slow but progressive decrease in the number of lymphocytes, particularly CD4+ cells, with age (17% reduction by 3 months, 58% reduction by 6 months). Allogeneic responses were then evaluated in vitro as well as in vivo using a subcutaneous sponge matrix implant. Although spleen cells cultured for 4 days in vitro with alloantigen developed normal functional responses, allogeneic responses generated in vivo within a subcutaneous sponge matrix were impaired. This was characterized by a depression in cytotoxic T lymphocyte (CTL) activity, a 82% reduction in the frequency of helper T cells, and a 78% reduction in the capacity of sponge-infiltrating lymphocytes to produce IL-2 in response to secondary antigen stimulation. These results indicate that although overt lymphocyte development and in vitro function were unremarkable, expression of a truncated ZAP-70 affected the in vivo survival of peripheral lymphocytes and altered the in vivo generation of functional activity to alloantigen.


Subject(s)
Protein-Tyrosine Kinases/physiology , T-Lymphocytes/immunology , Animals , Immunophenotyping , Interleukin-2/biosynthesis , Mice , Mice, Transgenic , Protein-Tyrosine Kinases/genetics , ZAP-70 Protein-Tyrosine Kinase
14.
J Leukoc Biol ; 62(5): 577-80, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9365111

ABSTRACT

Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes, memory T lymphocytes, and natural killer (NK) cells in vitro. Its expression has been documented in disorders characterized by mononuclear cell infiltrates, suggesting that it may contribute to the inflammatory component of such diseases as atherosclerosis, multiple sclerosis, or rheumatoid arthritis. To prove a causal association, the in vivo properties of MCP-1 must be understood. Several lines of transgenic mice have been constructed to address this question. A transgenic line in which MCP-1 expression is controlled by the MMTV-LTR expressed high levels of MCP-1 in multiple organs but showed no evidence for monocyte infiltration. Instead, these mice were more susceptible to infection by the intracellular pathogens, Listeria monocytogenes and Mycobacterium tuberculosis. These mice had high serum levels of MCP-1, suggesting that their circulating monocytes may have been desensitized or that MCP-1 stimulated a Th2-dominant response. In contrast, another model in which MCP-1 expression was controlled by the insulin promoter demonstrated a monocytic infiltrate in pancreatic islets. These results indicate that MCP-1 expression at low levels in an anatomically confined area results in monocyte infiltration, suggesting that when properly expressed, MCP-1's in vitro properties are reproduced in vivo. This justifies the examination of MCP-1-deficient mice in disease models in order to explore MCP-1's role in pathogenesis.


Subject(s)
Chemokine CCL2/physiology , Animals , Chemokine CCL2/biosynthesis , Chemokine CCL2/genetics , Mice , Mice, Transgenic
15.
J Immunol ; 159(7): 3595-602, 1997 Oct 01.
Article in English | MEDLINE | ID: mdl-9317159

ABSTRACT

In this study, we have evaluated the role of specific chemotactic cytokines in leukocyte recruitment to s.c. tissue in response to TNF-alpha in vivo. Injection of TNF-alpha into s.c. air pouches led to a rapid, transient accumulation of leukocytes. Maximal accumulation of leukocytes in the air pouch was observed at between 2 and 4 h after injection of TNF-alpha. The cellular exudate comprised predominantly neutrophils, with smaller numbers of eosinophils and mononuclear phagocytes also being recruited. However, lymphocyte recruitment was not observed. TNF-alpha injection induced a time-dependent increase in the levels of immunoreactive macrophage inflammatory protein (MIP)-2, MIP-1alpha, and JE in the pouch exudate as well as increased steady-state mRNA levels of KC, MIP-2, MIP-1alpha, and JE in the tissue lining the s.c. pouch and of MIP-2, MIP-1alpha, and JE in the exudate cell population. Passive immunization with specific Abs directed against each of these chemokines significantly inhibited the accumulation of neutrophils, mononuclear phagocytes, and eosinophils in response to TNF-alpha. Taken together, these data demonstrate the existence of a chemokine network in vivo involving at least four individual chemokines that regulates recruitment of the major peripheral blood granulocytes and mononuclear phagocytes to s.c. sites during acute inflammation. To our knowledge, these data are also the first demonstration that the C-C chemokine JE is involved in neutrophil recruitment in a physiologic system in vivo.


Subject(s)
Cell Movement/drug effects , Cell Movement/immunology , Chemokines/physiology , Neutrophils/immunology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antibodies/administration & dosage , Chemokines/biosynthesis , Chemokines/chemical synthesis , Chemokines/genetics , Chemokines/immunology , Diffusion Chambers, Culture , Exudates and Transudates/cytology , Exudates and Transudates/immunology , Gene Expression Regulation/immunology , Immunization, Passive , Injections, Intraperitoneal , Male , Mice , Mice, Inbred BALB C , Neutrophils/physiology
16.
J Immunol ; 159(1): 401-8, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9200479

ABSTRACT

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes in vitro; however, its in vivo functions are poorly understood. To address this question, we constructed transgenic mice expressing MCP-1 controlled by an insulin promoter. These mice developed a chronic insulitic infiltrate composed of F4/80+ monocytes with minor populations of CD4+, CD8+, and B220+ cells. Despite persistent transgene expression, the insulitis never progressed, and blood glucose levels remained normal. Thus, MCP-1 alone is sufficient to elicit a monocytic infiltrate, but not to activate elicited cells. These results differ from those obtained with another transgenic model using the mouse mammary tumor virus long terminal repeat, in which mice expressed substantial MCP-1 in several organs but had no infiltrates. However, mice expressing both transgenes had minimal insulitis, indicating that high systemic levels of MCP-1 prevented monocytes from responding to local MCP-1. Thus, the ability of MCP-1 to elicit monocytic infiltration depends on its being expressed at low levels in an anatomically restricted area.


Subject(s)
Chemokine CCL2/biosynthesis , Islets of Langerhans/immunology , Monocytes/immunology , Pancreatic Diseases/immunology , Animals , Cell Movement , Chemokine CCL2/genetics , Gene Transfer Techniques , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Mice , Mice, Transgenic , Monocytes/pathology , Pancreatic Diseases/genetics , Pancreatic Diseases/metabolism
17.
Int Immunol ; 8(10): 1617-26, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8921442

ABSTRACT

In our previous work, DNase hypersensitivity mapping was used to identify an enhancer within the human CD8 alpha (hCD8 alpha) gene which allowed T cell-specific expression of a reporter construct in transiently transfected cell lines. To study the role of this intronic enhancer in vivo, transgenic mice were made using human CD8 genomic constructs. We found that while a 14 kb wild-type human CD8 alpha (WThCD8) genomic construct did not lead to expression in mature peripheral CD8+ T cells, this transgene was consistently expressed in small populations of T cells and B cells, and in a subset of mouse NK cells. While murine CD8 is not normally expressed on resting NK cells, expression of the human CD8 transgene on mouse NK cells is appropriate since CD8 is expressed on a subset of human NK cells. Deletion of the intronic enhancer resulted in a complete loss of transgene expression in most lines and a loss of expression only in NK cells in one line. Our results indicate, firstly, that cis-acting sequences within the 14 kb genomic fragment are sufficient for NK cell-specific expression. In addition, our results suggest that the enhancer may have dual roles in regulation of transgene expression. It may enhance general expression of the transgene and may also be required for NK cell-specific expression.


Subject(s)
CD8 Antigens/biosynthesis , CD8 Antigens/genetics , Gene Expression/genetics , Killer Cells, Natural/metabolism , T-Lymphocytes, Cytotoxic/metabolism , Animals , Humans , Killer Cells, Lymphokine-Activated/metabolism , Killer Cells, Natural/immunology , Mice , Mice, Transgenic , T-Lymphocytes, Cytotoxic/immunology , Transgenes/immunology
18.
J Exp Med ; 183(4): 1893-8, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8666945

ABSTRACT

Leukotriene B4 (LTB4) is a chemotactic and cell-activating factor present at inflammatory sites in a variety of autoimmune diseases including multiple sclerosis (MS). In this study, we used a murine model of MS, experimental allergic encephalomyelitis (EAE), to assess the potential role of LTB4 on cell infiltration and paralysis. Injection of encephalogenic T cells into naive animals induced paralysis and weight loss that was completely inhibited by treatment with the selective LTB4 receptor antagonist CP-105,696 (ED50= 8.6 mg/kg orally). Although migration of lymphocytes into the central nervous system was unaffected, the efficacious effects of CP-105,696 correlated with up to a 97% decrease in eosinophil infiltration into the lower spinal cord as determined by light and electron microscopy and quantitated by levels of the specific enzyme marker eosinophil peroxidase. These results demonstrate that eosinophil recruitment in EAE is dependent on LTB4 receptor ligation and further reveal a previously unrecognized role for eosinophils in the pathogenesis of this disease.


Subject(s)
Benzopyrans/pharmacology , Carboxylic Acids/pharmacology , Cell Movement/drug effects , Encephalomyelitis, Autoimmune, Experimental/etiology , Eosinophils/drug effects , Receptors, Leukotriene B4/antagonists & inhibitors , Amino Acid Sequence , Animals , Benzopyrans/therapeutic use , Carboxylic Acids/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Female , Immunization, Passive , Mice , Mice, Inbred Strains , Molecular Sequence Data , Oligopeptides/immunology , Paralysis/prevention & control , Spinal Cord/pathology , T-Lymphocytes/immunology
19.
Antimicrob Agents Chemother ; 40(2): 314-19, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8834872

ABSTRACT

The activity of trovafloxacin against 22 clinical Legionella isolates was determined by broth microdilution susceptibility testing. The trovafloxacin concentration required to inhibit 90% of strains tested was < or = 0.004 micrograms/ml, in contrast to 0.032 micrograms/ml for ofloxacin. In guinea pig alveolar macrophages, trovafloxacin achieved intracellular levels up to 28-fold over the extracellular concentration, which was similar to the levels obtained with erythromycin. Trovafloxacin (0.25 micrograms/ml) reduced bacterial counts of two L. pneumophila strains grown in guinea pig alveolar macrophages by > 2 log10 CFU/ml, without regrowth, under drug-free conditions over a 3-day period; trovafloxacin was significantly more active than ofloxacin or erythromycin (0.25 to 1 microgram/ml) in this assay. Single-dose (10 mg of prodrug CP-116,517-27 per kg of body weight given intraperitoneally [i.p.], equivalent to 7.5 mg of trovafloxacin per kg) pharmacokinetic studies performed in guinea pigs with L. pneumophila pneumonia revealed peak serum and lung trovafloxacin levels to be 3.8 micrograms/ml and 5.0 micrograms/g, respectively, at 0.5 h and 4.2 micrograms/ml and 2.9 micrograms/g, respectively, at 1 h. Administration of a lower prodrug dose (1.4 mg of trovafloxacin equivalent per kg i.p.) gave levels in lung and serum of 0.4 microgram/g and 0.4 microgram/ml, respectively, 1 h after drug administration. The terminal half-lives of elimination from serum and lung were 0.8 and 1.1 h, respectively. All 15 infected guinea pigs treated for 5 days with CP-116,517-27 once daily (10 mg/kg/day i.p., equivalent to 7.5 mg of trovafloxacin per kg/day) survived for 10 days after antimicrobial therapy, as did all 15 guinea pigs treated with ofloxacin once daily (10 mg/kg/day i.p.) for 5 days. None of 13 animals treated with saline survived. In a second experiment with animals, trovafloxacin (1.4 mg/kg/day i.p. for 5 days) protected all 16 guinea pigs from death, whereas all 15 animals treated with saline died. Trovafloxacin is an effective antimicrobial agent against Legionella in vitro and in vivo, with the ability to concentrate in macrophages and kill intracellular organisms.


Subject(s)
Anti-Infective Agents/pharmacology , Fluoroquinolones , Legionella/drug effects , Legionellosis/metabolism , Macrophages, Alveolar/microbiology , Naphthyridines/pharmacology , Naphthyridines/pharmacokinetics , Prodrugs/pharmacokinetics , Animals , Anti-Bacterial Agents/pharmacology , Erythromycin/pharmacology , Guinea Pigs , Half-Life , Legionella/growth & development , Legionella pneumophila/drug effects , Legionellosis/drug therapy , Male , Microbial Sensitivity Tests , Naphthyridines/therapeutic use
20.
J Immunol ; 155(10): 4838-43, 1995 Nov 15.
Article in English | MEDLINE | ID: mdl-7594486

ABSTRACT

We have constructed transgenic mice in which the mouse mammary tumor virus long terminal repeat controls the expression of murine monocyte chemoattractant protein-1 (MCP-1). Several independently derived lines of transgenic mice constitutively expressed MCP-1 protein in a variety of organs. Protein extracts from these organs had substantial in vitro monocyte chemoattractant activity that was neutralized by an anti-MCP-1 Ab, indicating that transgenic MCP-1 protein is biologically active. However, no transgenic mouse at any age displayed monocyte infiltrates in MCP-1-expressing organs. Two transgenic lines had circulating MCP-1 levels of 13 to 26 ng/ml, which is a concentration sufficient to induce maximal monocyte chemotaxis in vitro. These transgenic lines showed a 1 to 1.5 log greater sensitivity to infection with Listeria monocytogenes and Mycobacterium tuberculosis. A third transgenic line had lower serum levels of MCP-1 and was resistant to L. monocytogenes. The results suggest that this transgenic model is one of monocyte nonresponsiveness to locally produced MCP-1 due to either receptor desensitization or neutralization of a chemoattractant gradient by high systemic concentrations of MCP-1. Regardless of the mechanism, the data indicate that constitutively high levels of MCP-1 expression do not induce monocytic infiltrates, and that MCP-1 is involved in the host response to intracellular pathogens.


Subject(s)
Chemokine CCL2/biosynthesis , Immune Tolerance , Listeria monocytogenes/immunology , Listeriosis/immunology , Monocytes/immunology , Mycobacterium tuberculosis/immunology , Tuberculosis/immunology , Animals , Cell Movement , Chemokine CCL2/genetics , Chemokine CCL2/immunology , Chemotaxis , Listeria monocytogenes/pathogenicity , Listeriosis/pathology , Mice , Mice, Transgenic , Mycobacterium tuberculosis/pathogenicity , Tuberculosis/pathology
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