BALB/c mice are susceptible to infection with the visceralizing species of Leishmania, Leishmania... more BALB/c mice are susceptible to infection with the visceralizing species of Leishmania, Leishmania chagasi. The parasite load initially rises in the liver and spontaneously subsides, whereas parasite multiplication begins later and remains lower in the spleen. To investigate whether this organ-specific multiplication of L. chagasi correlates with localized immune responses, we compared cytokine production by splenic vs hepatic immune cells. Livers from infected mice contained granulomas harboring intracellular L. chagasi amastigotes, whereas few amastigotes were present in the spleen. FACS analysis granuloma cells showed granuloma lymphocytes expressed a memory/effector phenotype. Granuloma cells cultured in vitro produced IL-10 and IL-6 but no detectable IFN-gamma, IL-4 or IL-5. In contrast, splenocytes from the same animals secreted IFN-gamma, IL-4, IL-6, and IL-10. T cells were depleted from granuloma cells by immune lysis, and the results indicated that IL-10 and IL-6 were derive...
Murine Schistosomiasis mansoni is a parasitic disease in which granulomas develop around the schi... more Murine Schistosomiasis mansoni is a parasitic disease in which granulomas develop around the schistosome ova that lodge in the liver and intestines of the host. The granuloma eosinophils make substance P (SP), a cytokine with immunoregulatory properties. Within the granuloma SP can modulate IFN-gamma production through interaction with a substance P-like receptor. SP belongs to a family of hormones called tachykinins. Three mammalian tachykinins are SP, neurokinin A (substance K), and neurokinin B (neuromedin K). In humans and rats, there are at least three distinct tachykinin receptors designated NK-1, NK-2, and NK-3. The NK-1 receptor binds only SP with high affinity. Using reverse transcription-PCR, cDNA cloning, and sequence analysis, we showed that granulomas isolated from the liver of infected mice express an authentic SP (NK-1) receptor but have no detectable neurokinin A (NK-2) and neurokinin B (NK-3) receptor mRNA, as determined by PCR. CD4+ granuloma T lymphocytes, purifie...
Tuberculosis (TB) is one of the most prevalent global health crises in the world today and is cau... more Tuberculosis (TB) is one of the most prevalent global health crises in the world today and is caused by the bacterium Mycobacterium Tuberculosis (Mtb). While CD4+ T cells are known to be critical in the protective response against Mtb, the importance of CD8+T cell responses are gaining attention. The current vaccine BCG has limited and variable efficacy. BCG induces a strong CD4, but weak CD8+ T cell response. Targeting epitopes to specific dendritic cell receptors is an efficient way to induce T cell immunity. We created recombinant antibodies that recognize the pro-phagocytic receptor DEC205 (CD205), the co-stimulatory molecule CD40, and the phagocytic/activating Fc receptors 2/3 (FcR II/III) (CD16/32). Fused to the Fc portion of the antibodies is an Mtb immunodominant peptide, as well as a CD8+ T cell epitope. Individually, or in combination, these antibodies induce robust CD4+, antigen specific immune response in vivo. Here, we report that these DC receptor-specific recombinant ...
Schistosomiasis mansoni is a disease characterized by liver and intestinal granulomas. Previous i... more Schistosomiasis mansoni is a disease characterized by liver and intestinal granulomas. Previous investigations in our laboratory showed that nylon wool-adherent CD4+ T lymphocytes isolated from murine hepatic schistosome granulomas express receptors for somatostatin 1-14. Moreover, somatostatin 1-14 substantially decreased IFN-gamma and IgG2a, but not IL-5 secretion by dispersed granuloma cells. This paper extends these observations by defining the somatostatin receptor (SSTR) isoform most likely expressed by granuloma inflammatory lymphocytes. Amplification of mRNA by reverse transcription PCR shows SSTR1, SSTR2, and SSTR3 mRNA in normal mouse brain and other tissues. Nevertheless, only the SSTR2 PCR product was amplified from granuloma cell RNA. The nucleotide sequence of the granuloma SSTR2 PCR product from inflammatory cells is identical to the CBA murine brain SSTR2 cDNA sequence. Granuloma-derived T cell lines, FACS-isolated granuloma CD4+ T cells, thymocytes, splenocytes, and...
CD4+ Th cell infiltration into the brain and the activation by cellular elements of the central n... more CD4+ Th cell infiltration into the brain and the activation by cellular elements of the central nervous system (CNS) are thought to be important steps in the initiation of CNS autoimmune diseases. T cell activation requires Ag-specific stimulation and additional costimulatory signals provided by the APC. Here we describe how murine brain microvessel endothelial (En) cells and smooth muscle/pericytes (SM/P) selectively induce the Ag-specific activation of different Th1 and Th2 CD4+ T cell clones. Th1 and Th2 cell clones were used that were specific for the same peptide Ag in the context of the same class II allotype. SM/P preferentially activated Th1 cell clones, whereas En cells activated Th2 cell clones better, as reflected by cell proliferation and production of IL-2 by SM/P-activated Th1 clones and IL-4 by Th2 clones. There was no difference in the level of expression of CD4, CD2, or LFA-1 molecules between these Th cell clones, and anti-CD4, CD2, LFA-1 or ICAM-1 mAb did not diff...
Granulomas form in the liver and intestines of mice infected with the parasite Schistosoma manson... more Granulomas form in the liver and intestines of mice infected with the parasite Schistosoma mansoni. Vasoactive intestinal peptide (VIP) is a neurokine that can modulate aspects of the immune response by acting through receptors within the granuloma. Cloned are two novel VIP receptor (VIPR) mRNAs (VIPR1 and VIPR2) that also bind a second neurokine called pituitary adenylated cyclase-activating polypeptide (PACAP). The objective of this study was to determine if granulomas express either VIPR1 or VIPR2. Using a radioligand-binding assay, it was established that PACAP is as effective as VIP at displacing radiolabeled VIP from splenocytes and granuloma cells, and that most if not all VIPRs in the spleen and granulomas bind PACAP. PCR amplification of reverse transcribed RNA determined that granulomas express both VIPR1 and VIPR2 mRNAs. Gel electrophoresis and nucleotide sequencing confirmed the authenticity of the PCR products. Also, both receptor subtypes were amplified from several gr...
Handbook of Neurochemistry and Molecular Neurobiology, 2008
ABSTRACT There is a growing body of evidence shedding light on the mechanism of immune regulation... more ABSTRACT There is a growing body of evidence shedding light on the mechanism of immune regulation in the central nervous system (CNS). There are multiple elements that contribute to the special anti-inflammatory environment in this tissue. Unique physiological and anatomic features, soluble mediators and cell surface molecules and immune cells promote the immune privilege status of the CNS. These multiple mechanisms together shape immune responses in the inflamed CNS. A deeper understanding the mechanism of immune privilege in the CNS will provide the basic of successful therapeutic interventions for immune-mediated CNS diseases, such as multiple sclerosis (MS).
Journal of Neuropathology & Experimental Neurology, 2001
Trafficking of antigen-specific T cells into the central nervous system (CNS) is an important ini... more Trafficking of antigen-specific T cells into the central nervous system (CNS) is an important initiating step in inflammation in the brain. In spite of the extensive knowledge about the role of adhesion molecules in T cell migration across peripheral vessels, the mechanism of the entry of antigen-specific T cells into the CNS is not known. This work was designed to study the regulatory roles of adhesion molecules in antigen-specific T cell migration into the CNS. Antigen-specific T cells were tracked in an in vivo migration assay using T cell receptor (TCR) transgenic mice having 95% of T cells specific for a defined antigen, pigeon cytochrome c (PCC). TCR transgenic mice were cannulated intraventricularly (IVT) for PCC antigen infusion and cerebrospinal fluid (CSF) sampling. Upon PCC infusion into the CNS, the number of ␣/ TCR ϩ V3 ϩ Mac1 Ϫ cells in the CSF was characterized in the presence or absence of anti-adhesion molecule reagents. We found that antibodies against VCAM-1 (CD106), VLA-4 (CD49d/CD29), ICAM-1 (CD54), and LFA-1 (CD11a/CD18) did not influence the increased number of antigen-specific T cells in the CSF. However, upon intravenous (IV) injection, anti-PECAM-1 (CD31) antibody or PECAM-Ig chimeric molecule inhibited the trafficking of ␣/ TCR ϩ V3 ϩ Mac1 Ϫ cells into the CNS. The expression of PECAM-1 (CD31) was also up-regulated on antigen-specific T cells in a time-dependent manner in vitro upon antigenic stimulation. The antigen-induced activation of T cells in vivo was measured by CD44 and LFA-1 expression and found to be comparable between mPECAMIg-treated mice and wild-type serum control-treated groups. This indicates that CD31 inhibition of antigen-specific T cell accumulation in the CNS is probably not due to a functional inhibition of these cells. Finally, adoptive transfer of CFSE-labeled AND transgenic cells into naï ve animals resulted in the accumulation of these cells in the CNS upon PCC IVT immunization that was also inhibited by mPECAMIg treatment. Hence, PECAM-1 (CD31) might play an important role in regulating antigen-specific T cells trafficking in CNS inflammatory diseases.
The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the Nε-lysine acetyla... more The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the Nε-lysine acetylation of ER-resident and ER-transiting proteins. A point-mutation (S113R) in AT-1 has been associated with a familial form of spastic paraplegia. Here, we report that AT-1S113Ris unable to form homodimers in the ER membrane and is devoid of acetyl-CoA transport activity. The reduced influx of acetyl-CoA into the ER lumen results in reduced acetylation of ER proteins and an aberrant form of autophagy. Mice homozygous for the mutation display early developmental arrest. In contrast, heterozygous animals develop to full term, but display neurodegeneration and propensity to infections, inflammation, and cancer. The immune and cancer phenotypes are contingent on the presence of pathogens in the colony, whereas the nervous system phenotype is not. In conclusion, our results reveal a previously unknown aspect of acetyl-CoA metabolism that affects the immune and nervous systems and the risk for m...
The plasma membrane of rat peritoneal exudate cells was spin labelled with fatty acid spin probes... more The plasma membrane of rat peritoneal exudate cells was spin labelled with fatty acid spin probes and structural changes induced by ligand binding to Fc and C3b receptors were followed. Neither antigens, nor monovalent immune complexes were capable of inducing any change. Multivalent ligands, both in soluble and particulate form, and to a lesser extent uncomplexed immunoglobulins were very effective in inducing membrane reordering , i.e. a decrease in fluidity. The phase transition region markedly broadened upon binding of multivalent immune complexes, indicating phase separation. There was a time lag of S-10 min before the onset of lipid reordering was observed. We suggest that multivalent binding that iscapable of cross-linking surface receptors initiates lateral phase separation and lipid reordering due to receptor aggregation.
The macrophage plasma membrane was labelled with an intercalated S-doxyl stearic acid spin probe,... more The macrophage plasma membrane was labelled with an intercalated S-doxyl stearic acid spin probe, and structural changes induced by IgG-coated erythrocytes (EA) were followed with particular emphasis on the possible role of lipid reordering in the sequential events of phagocytosis. We present three lines of experimental evidence to show that these structural changes were induced by the lateral aggregation of cell surface Fc receptors. Cytochalasin B. an inhibitor of microfilament functton. blocked this membrane reordering; if it was added after EA binding induced membrane reordering had already been detected for I5 min. a rapid reversal process was observed resulting in a reversible restoration of the initial order parameter value. We suggest that these structural changes indicate lipid-lipid lateral phase separation.
Based on the observation that the application of monoclonal antibodies (mAbs) directed against ct... more Based on the observation that the application of monoclonal antibodies (mAbs) directed against ct4-integrin and VCAM-I inhibit the development of experimental autoimmune encephalomyelitis (EAE) in vivo it has been concluded that the
dase staining of frozen sections with anti-cytokine mAbs localized IL-2 and IFN-y production in C... more dase staining of frozen sections with anti-cytokine mAbs localized IL-2 and IFN-y production in CNS to perivascular infiltrating CD4 + T cells. CD4 ÷ T cells were isolated from EAE CNS by discontinuous Percoll centrifugation and analyzed by flow cytometry. All were CD44 high, and the majority were CD45RB l°w, a memory/effector phenotype that has been associated with elevated cytokine production in vitro. The number of mononuclear infiltrates in spinal cords from animals that had remitted a week earlier and showed no symptoms of EAE was markedly reduced. The number of CD4 ÷ T cells in CNS mononuclear isolates from these animals was only slightly greater than in unprimed animals, and very much reduced from peak disease. The levels of CD3, IL-2 and IFN-y mRNA in the CNS of animals in remission were indistinguishable from controls. These results suggest that CD3 ÷ T cells leave the CNS during remission, thereby abrogating cytokine production in the CNS. The mononuclear cells which remain in the CNS in remission, and their cytokine output (if any), remain to be identified.
The activation of Ag-specific T cells locally in the CNS could potentially contribute to the deve... more The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8+ T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c+ cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c+ cells stimulate Ag-specific naive CD8+ T cells locally in the CNS and may contribute to immune responses in the brain.
A panel of 20 murine CD4+ clones was examined for the presence of surface membrane receptors for ... more A panel of 20 murine CD4+ clones was examined for the presence of surface membrane receptors for IgA, IgM, IgD, IgE, and IgG. High level expression of multiple Fc receptors (FcRs) was found on all Th2 clones. FcR expression was low or undetected on the Th1 clones. The preferential expression of FcR on activated Th2 cells suggests potential mechanisms for immunoregulatory interactions with B cells.
Progenitor cells of the T- and B-lineages in mice express (CD32) and FcγRIII (CD16) but as the de... more Progenitor cells of the T- and B-lineages in mice express (CD32) and FcγRIII (CD16) but as the developing lymphocytes begin to express clonal antigen receptors, CD16 and CD32 are downregulated in T-cells, and CD16 is downregulated in B-cells. Considering that counter-receptors for FcγR occur on thymic and bone marrow stromal cells, the possibility exists that FcγR might participate in some
CD23, a low-affinity IgE Fc receptor, is not displayed on most resting T cells but its expression... more CD23, a low-affinity IgE Fc receptor, is not displayed on most resting T cells but its expression has been shown to be transiently induced in vivo and in vitro on some CD4+ T cells [1-4] and in vivo on CD8+ T cells by IgE-secreting hybridoma tumors [5]. To investigate the functional role of CD23 on T cells, we inserted a CD23 construct into an expression vector driven by a CD2 promoter and transfected it into a murine Th2 clone D10.G4.1 (D10). We stimulated the transfected D10 cells (D10.3M.24) with anti-TCR antibody in the presence or absence of IgE, and measured IL-4, IL-5 and IL-6 production in the culture supernatants. Activation of D10.3M.24 cells by anti-TCR antibody induced greater levels of IL-4, IL-5 and IL-6 production, when the TCR and CD23 were co-crosslinked by TNP anti-TCR and IgE anti-TNP antibodies. IgG anti-TNP antibody did not enhance lymphokine production by D10.3M.24 cells. The enhanced lymphokine production by IgE was blocked by monoclonal anti-CD23 antibody. IgE anti-TNP antibody did not enhance lymphokine production by the wild-type D10 cells induced by TNP anti-TCR antibody. These studies show that when co-crosslinked with the TCR, CD23 can modulate the lymphokine production in activated Th2 cells. Since CD23 binds to IgE and also binds to CD21 [6], a complement receptor commonly expressed on B cells, T-cell CD23 could play an immunoregulatory role during cognate T-B cell interaction and during IgE antibody responses.
γδ T cells have a crucial role in cell-mediated immunity (CMI) against P. chabaudi malaria, but δ... more γδ T cells have a crucial role in cell-mediated immunity (CMI) against P. chabaudi malaria, but δ-chain knockout (KO) (δo/o) mice and mice depleted of γδ T cells with mAb cure this infection. To address the question of why mice deficient in γδ T cells resolve P. chabaudi infections, we immunized δo/o mice by infection with viable blood-stage parasites. Sera from infection-immunized mice were tested for their ability to protect JHo/o, δo/o double KO mice passively against P. chabaudi challenge infection. The onset of parasitemia was significantly delayed in mice receiving immune sera, compared with saline or uninfected serum controls. Immune sera were then fractionated into Ig-rich and Ig-depleted fractions by HPLC on a protein G column. Double KO mice were passively immunized with either fraction and challenged with P. chabaudi. The onset of parasitemia was significantly delayed in recipients of the Ig-rich fraction compared with recipients of the Ig-poor fraction of immune sera. We...
Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation as... more Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation associated with autoimmune disorders, such as multiple sclerosis, but the contribution of these cells to the outcome of disease is not yet clear. Here, we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigen-specific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE, accompanied with an early T-cell infiltration and a decreased proportion of regulatory FoxP3-expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor α induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific ...
BALB/c mice are susceptible to infection with the visceralizing species of Leishmania, Leishmania... more BALB/c mice are susceptible to infection with the visceralizing species of Leishmania, Leishmania chagasi. The parasite load initially rises in the liver and spontaneously subsides, whereas parasite multiplication begins later and remains lower in the spleen. To investigate whether this organ-specific multiplication of L. chagasi correlates with localized immune responses, we compared cytokine production by splenic vs hepatic immune cells. Livers from infected mice contained granulomas harboring intracellular L. chagasi amastigotes, whereas few amastigotes were present in the spleen. FACS analysis granuloma cells showed granuloma lymphocytes expressed a memory/effector phenotype. Granuloma cells cultured in vitro produced IL-10 and IL-6 but no detectable IFN-gamma, IL-4 or IL-5. In contrast, splenocytes from the same animals secreted IFN-gamma, IL-4, IL-6, and IL-10. T cells were depleted from granuloma cells by immune lysis, and the results indicated that IL-10 and IL-6 were derive...
Murine Schistosomiasis mansoni is a parasitic disease in which granulomas develop around the schi... more Murine Schistosomiasis mansoni is a parasitic disease in which granulomas develop around the schistosome ova that lodge in the liver and intestines of the host. The granuloma eosinophils make substance P (SP), a cytokine with immunoregulatory properties. Within the granuloma SP can modulate IFN-gamma production through interaction with a substance P-like receptor. SP belongs to a family of hormones called tachykinins. Three mammalian tachykinins are SP, neurokinin A (substance K), and neurokinin B (neuromedin K). In humans and rats, there are at least three distinct tachykinin receptors designated NK-1, NK-2, and NK-3. The NK-1 receptor binds only SP with high affinity. Using reverse transcription-PCR, cDNA cloning, and sequence analysis, we showed that granulomas isolated from the liver of infected mice express an authentic SP (NK-1) receptor but have no detectable neurokinin A (NK-2) and neurokinin B (NK-3) receptor mRNA, as determined by PCR. CD4+ granuloma T lymphocytes, purifie...
Tuberculosis (TB) is one of the most prevalent global health crises in the world today and is cau... more Tuberculosis (TB) is one of the most prevalent global health crises in the world today and is caused by the bacterium Mycobacterium Tuberculosis (Mtb). While CD4+ T cells are known to be critical in the protective response against Mtb, the importance of CD8+T cell responses are gaining attention. The current vaccine BCG has limited and variable efficacy. BCG induces a strong CD4, but weak CD8+ T cell response. Targeting epitopes to specific dendritic cell receptors is an efficient way to induce T cell immunity. We created recombinant antibodies that recognize the pro-phagocytic receptor DEC205 (CD205), the co-stimulatory molecule CD40, and the phagocytic/activating Fc receptors 2/3 (FcR II/III) (CD16/32). Fused to the Fc portion of the antibodies is an Mtb immunodominant peptide, as well as a CD8+ T cell epitope. Individually, or in combination, these antibodies induce robust CD4+, antigen specific immune response in vivo. Here, we report that these DC receptor-specific recombinant ...
Schistosomiasis mansoni is a disease characterized by liver and intestinal granulomas. Previous i... more Schistosomiasis mansoni is a disease characterized by liver and intestinal granulomas. Previous investigations in our laboratory showed that nylon wool-adherent CD4+ T lymphocytes isolated from murine hepatic schistosome granulomas express receptors for somatostatin 1-14. Moreover, somatostatin 1-14 substantially decreased IFN-gamma and IgG2a, but not IL-5 secretion by dispersed granuloma cells. This paper extends these observations by defining the somatostatin receptor (SSTR) isoform most likely expressed by granuloma inflammatory lymphocytes. Amplification of mRNA by reverse transcription PCR shows SSTR1, SSTR2, and SSTR3 mRNA in normal mouse brain and other tissues. Nevertheless, only the SSTR2 PCR product was amplified from granuloma cell RNA. The nucleotide sequence of the granuloma SSTR2 PCR product from inflammatory cells is identical to the CBA murine brain SSTR2 cDNA sequence. Granuloma-derived T cell lines, FACS-isolated granuloma CD4+ T cells, thymocytes, splenocytes, and...
CD4+ Th cell infiltration into the brain and the activation by cellular elements of the central n... more CD4+ Th cell infiltration into the brain and the activation by cellular elements of the central nervous system (CNS) are thought to be important steps in the initiation of CNS autoimmune diseases. T cell activation requires Ag-specific stimulation and additional costimulatory signals provided by the APC. Here we describe how murine brain microvessel endothelial (En) cells and smooth muscle/pericytes (SM/P) selectively induce the Ag-specific activation of different Th1 and Th2 CD4+ T cell clones. Th1 and Th2 cell clones were used that were specific for the same peptide Ag in the context of the same class II allotype. SM/P preferentially activated Th1 cell clones, whereas En cells activated Th2 cell clones better, as reflected by cell proliferation and production of IL-2 by SM/P-activated Th1 clones and IL-4 by Th2 clones. There was no difference in the level of expression of CD4, CD2, or LFA-1 molecules between these Th cell clones, and anti-CD4, CD2, LFA-1 or ICAM-1 mAb did not diff...
Granulomas form in the liver and intestines of mice infected with the parasite Schistosoma manson... more Granulomas form in the liver and intestines of mice infected with the parasite Schistosoma mansoni. Vasoactive intestinal peptide (VIP) is a neurokine that can modulate aspects of the immune response by acting through receptors within the granuloma. Cloned are two novel VIP receptor (VIPR) mRNAs (VIPR1 and VIPR2) that also bind a second neurokine called pituitary adenylated cyclase-activating polypeptide (PACAP). The objective of this study was to determine if granulomas express either VIPR1 or VIPR2. Using a radioligand-binding assay, it was established that PACAP is as effective as VIP at displacing radiolabeled VIP from splenocytes and granuloma cells, and that most if not all VIPRs in the spleen and granulomas bind PACAP. PCR amplification of reverse transcribed RNA determined that granulomas express both VIPR1 and VIPR2 mRNAs. Gel electrophoresis and nucleotide sequencing confirmed the authenticity of the PCR products. Also, both receptor subtypes were amplified from several gr...
Handbook of Neurochemistry and Molecular Neurobiology, 2008
ABSTRACT There is a growing body of evidence shedding light on the mechanism of immune regulation... more ABSTRACT There is a growing body of evidence shedding light on the mechanism of immune regulation in the central nervous system (CNS). There are multiple elements that contribute to the special anti-inflammatory environment in this tissue. Unique physiological and anatomic features, soluble mediators and cell surface molecules and immune cells promote the immune privilege status of the CNS. These multiple mechanisms together shape immune responses in the inflamed CNS. A deeper understanding the mechanism of immune privilege in the CNS will provide the basic of successful therapeutic interventions for immune-mediated CNS diseases, such as multiple sclerosis (MS).
Journal of Neuropathology & Experimental Neurology, 2001
Trafficking of antigen-specific T cells into the central nervous system (CNS) is an important ini... more Trafficking of antigen-specific T cells into the central nervous system (CNS) is an important initiating step in inflammation in the brain. In spite of the extensive knowledge about the role of adhesion molecules in T cell migration across peripheral vessels, the mechanism of the entry of antigen-specific T cells into the CNS is not known. This work was designed to study the regulatory roles of adhesion molecules in antigen-specific T cell migration into the CNS. Antigen-specific T cells were tracked in an in vivo migration assay using T cell receptor (TCR) transgenic mice having 95% of T cells specific for a defined antigen, pigeon cytochrome c (PCC). TCR transgenic mice were cannulated intraventricularly (IVT) for PCC antigen infusion and cerebrospinal fluid (CSF) sampling. Upon PCC infusion into the CNS, the number of ␣/ TCR ϩ V3 ϩ Mac1 Ϫ cells in the CSF was characterized in the presence or absence of anti-adhesion molecule reagents. We found that antibodies against VCAM-1 (CD106), VLA-4 (CD49d/CD29), ICAM-1 (CD54), and LFA-1 (CD11a/CD18) did not influence the increased number of antigen-specific T cells in the CSF. However, upon intravenous (IV) injection, anti-PECAM-1 (CD31) antibody or PECAM-Ig chimeric molecule inhibited the trafficking of ␣/ TCR ϩ V3 ϩ Mac1 Ϫ cells into the CNS. The expression of PECAM-1 (CD31) was also up-regulated on antigen-specific T cells in a time-dependent manner in vitro upon antigenic stimulation. The antigen-induced activation of T cells in vivo was measured by CD44 and LFA-1 expression and found to be comparable between mPECAMIg-treated mice and wild-type serum control-treated groups. This indicates that CD31 inhibition of antigen-specific T cell accumulation in the CNS is probably not due to a functional inhibition of these cells. Finally, adoptive transfer of CFSE-labeled AND transgenic cells into naï ve animals resulted in the accumulation of these cells in the CNS upon PCC IVT immunization that was also inhibited by mPECAMIg treatment. Hence, PECAM-1 (CD31) might play an important role in regulating antigen-specific T cells trafficking in CNS inflammatory diseases.
The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the Nε-lysine acetyla... more The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the Nε-lysine acetylation of ER-resident and ER-transiting proteins. A point-mutation (S113R) in AT-1 has been associated with a familial form of spastic paraplegia. Here, we report that AT-1S113Ris unable to form homodimers in the ER membrane and is devoid of acetyl-CoA transport activity. The reduced influx of acetyl-CoA into the ER lumen results in reduced acetylation of ER proteins and an aberrant form of autophagy. Mice homozygous for the mutation display early developmental arrest. In contrast, heterozygous animals develop to full term, but display neurodegeneration and propensity to infections, inflammation, and cancer. The immune and cancer phenotypes are contingent on the presence of pathogens in the colony, whereas the nervous system phenotype is not. In conclusion, our results reveal a previously unknown aspect of acetyl-CoA metabolism that affects the immune and nervous systems and the risk for m...
The plasma membrane of rat peritoneal exudate cells was spin labelled with fatty acid spin probes... more The plasma membrane of rat peritoneal exudate cells was spin labelled with fatty acid spin probes and structural changes induced by ligand binding to Fc and C3b receptors were followed. Neither antigens, nor monovalent immune complexes were capable of inducing any change. Multivalent ligands, both in soluble and particulate form, and to a lesser extent uncomplexed immunoglobulins were very effective in inducing membrane reordering , i.e. a decrease in fluidity. The phase transition region markedly broadened upon binding of multivalent immune complexes, indicating phase separation. There was a time lag of S-10 min before the onset of lipid reordering was observed. We suggest that multivalent binding that iscapable of cross-linking surface receptors initiates lateral phase separation and lipid reordering due to receptor aggregation.
The macrophage plasma membrane was labelled with an intercalated S-doxyl stearic acid spin probe,... more The macrophage plasma membrane was labelled with an intercalated S-doxyl stearic acid spin probe, and structural changes induced by IgG-coated erythrocytes (EA) were followed with particular emphasis on the possible role of lipid reordering in the sequential events of phagocytosis. We present three lines of experimental evidence to show that these structural changes were induced by the lateral aggregation of cell surface Fc receptors. Cytochalasin B. an inhibitor of microfilament functton. blocked this membrane reordering; if it was added after EA binding induced membrane reordering had already been detected for I5 min. a rapid reversal process was observed resulting in a reversible restoration of the initial order parameter value. We suggest that these structural changes indicate lipid-lipid lateral phase separation.
Based on the observation that the application of monoclonal antibodies (mAbs) directed against ct... more Based on the observation that the application of monoclonal antibodies (mAbs) directed against ct4-integrin and VCAM-I inhibit the development of experimental autoimmune encephalomyelitis (EAE) in vivo it has been concluded that the
dase staining of frozen sections with anti-cytokine mAbs localized IL-2 and IFN-y production in C... more dase staining of frozen sections with anti-cytokine mAbs localized IL-2 and IFN-y production in CNS to perivascular infiltrating CD4 + T cells. CD4 ÷ T cells were isolated from EAE CNS by discontinuous Percoll centrifugation and analyzed by flow cytometry. All were CD44 high, and the majority were CD45RB l°w, a memory/effector phenotype that has been associated with elevated cytokine production in vitro. The number of mononuclear infiltrates in spinal cords from animals that had remitted a week earlier and showed no symptoms of EAE was markedly reduced. The number of CD4 ÷ T cells in CNS mononuclear isolates from these animals was only slightly greater than in unprimed animals, and very much reduced from peak disease. The levels of CD3, IL-2 and IFN-y mRNA in the CNS of animals in remission were indistinguishable from controls. These results suggest that CD3 ÷ T cells leave the CNS during remission, thereby abrogating cytokine production in the CNS. The mononuclear cells which remain in the CNS in remission, and their cytokine output (if any), remain to be identified.
The activation of Ag-specific T cells locally in the CNS could potentially contribute to the deve... more The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8+ T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c+ cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c+ cells stimulate Ag-specific naive CD8+ T cells locally in the CNS and may contribute to immune responses in the brain.
A panel of 20 murine CD4+ clones was examined for the presence of surface membrane receptors for ... more A panel of 20 murine CD4+ clones was examined for the presence of surface membrane receptors for IgA, IgM, IgD, IgE, and IgG. High level expression of multiple Fc receptors (FcRs) was found on all Th2 clones. FcR expression was low or undetected on the Th1 clones. The preferential expression of FcR on activated Th2 cells suggests potential mechanisms for immunoregulatory interactions with B cells.
Progenitor cells of the T- and B-lineages in mice express (CD32) and FcγRIII (CD16) but as the de... more Progenitor cells of the T- and B-lineages in mice express (CD32) and FcγRIII (CD16) but as the developing lymphocytes begin to express clonal antigen receptors, CD16 and CD32 are downregulated in T-cells, and CD16 is downregulated in B-cells. Considering that counter-receptors for FcγR occur on thymic and bone marrow stromal cells, the possibility exists that FcγR might participate in some
CD23, a low-affinity IgE Fc receptor, is not displayed on most resting T cells but its expression... more CD23, a low-affinity IgE Fc receptor, is not displayed on most resting T cells but its expression has been shown to be transiently induced in vivo and in vitro on some CD4+ T cells [1-4] and in vivo on CD8+ T cells by IgE-secreting hybridoma tumors [5]. To investigate the functional role of CD23 on T cells, we inserted a CD23 construct into an expression vector driven by a CD2 promoter and transfected it into a murine Th2 clone D10.G4.1 (D10). We stimulated the transfected D10 cells (D10.3M.24) with anti-TCR antibody in the presence or absence of IgE, and measured IL-4, IL-5 and IL-6 production in the culture supernatants. Activation of D10.3M.24 cells by anti-TCR antibody induced greater levels of IL-4, IL-5 and IL-6 production, when the TCR and CD23 were co-crosslinked by TNP anti-TCR and IgE anti-TNP antibodies. IgG anti-TNP antibody did not enhance lymphokine production by D10.3M.24 cells. The enhanced lymphokine production by IgE was blocked by monoclonal anti-CD23 antibody. IgE anti-TNP antibody did not enhance lymphokine production by the wild-type D10 cells induced by TNP anti-TCR antibody. These studies show that when co-crosslinked with the TCR, CD23 can modulate the lymphokine production in activated Th2 cells. Since CD23 binds to IgE and also binds to CD21 [6], a complement receptor commonly expressed on B cells, T-cell CD23 could play an immunoregulatory role during cognate T-B cell interaction and during IgE antibody responses.
γδ T cells have a crucial role in cell-mediated immunity (CMI) against P. chabaudi malaria, but δ... more γδ T cells have a crucial role in cell-mediated immunity (CMI) against P. chabaudi malaria, but δ-chain knockout (KO) (δo/o) mice and mice depleted of γδ T cells with mAb cure this infection. To address the question of why mice deficient in γδ T cells resolve P. chabaudi infections, we immunized δo/o mice by infection with viable blood-stage parasites. Sera from infection-immunized mice were tested for their ability to protect JHo/o, δo/o double KO mice passively against P. chabaudi challenge infection. The onset of parasitemia was significantly delayed in mice receiving immune sera, compared with saline or uninfected serum controls. Immune sera were then fractionated into Ig-rich and Ig-depleted fractions by HPLC on a protein G column. Double KO mice were passively immunized with either fraction and challenged with P. chabaudi. The onset of parasitemia was significantly delayed in recipients of the Ig-rich fraction compared with recipients of the Ig-poor fraction of immune sera. We...
Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation as... more Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation associated with autoimmune disorders, such as multiple sclerosis, but the contribution of these cells to the outcome of disease is not yet clear. Here, we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigen-specific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE, accompanied with an early T-cell infiltration and a decreased proportion of regulatory FoxP3-expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor α induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific ...
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Papers by M. Sandor