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C L Baldwin

Publications and source records attributed to C L Baldwin.

At least 37 records · Page 2Linked to original sources
PubMed1995

Interleukin-10 downregulates protective immunity to Brucella abortus.

In vivo neutralization of interleukin-10 (IL-10) with an anti-IL-10 monoclonal antibody resulted in up to 10-fold fewer bacteria in the spleens of BALB/c mice infected with the virulent Brucella abortus strain 2308. In vitro neutralization of endogenous IL-10 in brucella antigen-stimulated cultures of splenocytes from infected mice resulted in increased gamma interferon production in these cultures, whereas exogenous recombinant IL-10 inhibited the ability of peptone-elicited peritoneal macrophages to control intracellular brucellae. These data suggest that IL-10 may be downregulating the immune response to B. abortus by affecting both macrophage effector function and the production of the protective Th1 cytokine gamma interferon.

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PubMed1995

Monocytes control gamma/delta T-cell responses by a secreted product.

Gamma-irradiated ex vivo bovine monocytes induce proliferation of gamma/delta T cells in the autologous mixed lymphocyte reaction (AMLR), whereas when not irradiated they prevent this response. In contrast, non-irradiated autologous monocytes have no effect on bovine alpha/beta T-cell proliferation in the allogenic MLR suggesting that the regulation is specific for gamma/delta T-cell responses. Here, we showed that the inhibition was not mediated by inducing cell death and that the ability of ex vivo monocytes to prevent proliferation of gamma/delta T cells was not generalized in that gamma/delta T cells still responded to mitogenic stimulation. Inhibition of the AMLR by non-irradiated monocytes could not be overcome by addition of interleukin-2 to the cultures or by costimulation with antibodies to WC1, a gamma/delta T-cell-specific cell-surface differentiation antigen shown elsewhere by us to be involved in activation of gamma/delta T cells. Furthermore, we showed that monocytes inhibited gamma/delta T-cell responses via a soluble product since inhibition occurred even when monocytes and gamma/delta T cells were separated by membranes of transwells or when supernatants from monocyte cultures were added to AMLR cultures. Maximal secretion of the inhibitory product by the monocytes occurred during the first 6 hr of in vitro culture at 37 degrees, rapidly decreased thereafter, and did not occur when monocytes were incubated at 4 degrees. The inhibition was not attributable to nitric oxide, reactive oxygen intermediates, prostaglandin E2 or transforming growth factor-beta (TGF-beta) but the ability of monocyte supernatants to mediate inhibition was sensitive to heating at 65 degrees. Lipopolysaccharide and granulocyte-macrophage colony-stimulating factor activation of monocytes temporarily abrogated their ability to inhibit proliferation. In contrast, heat-shocking had no effect on their ability to inhibit. We hypothesize that non-irradiated monocytes produce the inhibitory material in vivo in order to regulate gamma/delta T-cell responses to self-derived monocyte membrane components, but that when monocytes are altered by infection, transformation, irradiation, or cytokine activation, production of the inhibitor is temporarily suspended allowing stimulation of gamma/delta T cells to occur.

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PubMed1994

Characterization of resting and phorbol ester or concanavalin A activated bovine lymph node cells with leukocyte specific monoclonal antibodies.

Bovine lymph node cells (LNC) have been used as a model to study cell activation and proliferation. Because monoclonal antibodies to bovine lymphoid-specific surface antigens have only recently become available, these cells have not been previously characterized in regard to subpopulations. Furthermore, it was not known how expression of lymphoid differentiation antigens and subset proportionalities might change following different modes of activation of LNC. Therefore, the distribution of cell-surface differentiation antigens in unstimulated LNC as well as in LNC incubated with the mitogen concanavalin A (Con A) or the phorbol ester, phorbol dibutyrate (PDBU), was measured using a series of leukocyte-specific monoclonal antibodies and flow cytometry. Unstimulated LNC were found to have similar proportions of T cells, B cells (sIgM positive), and MHC Class II positive cells similar to bovine peripheral blood mononuclear leukocytes (reviewed by Baldwin et al., 1988a). Treatment of the LNC with PDBU or mitogenic doses of Con A induced changes in the expression of surface antigens consistent with the changes observed with human and mouse cells after similar activation. However, these two compounds did not cause identical effects. After treatment with PDBU, the percentage of cells expressing CD4 as well as the density of surface expression decreased. An increase in the percentage of cells expressing and/or density of surface expression of the pan T cell antigens CD2, CD5, CD6, MHC Class II and J5, a T cell activation antigen, also occurred. PDBU treatment also increased the percentage of CD8 positive cells. The change in CD6 following PDBU treatment has not been reported previously. Con A treatment led to a significant increase in the percentage of cells bearing CD8, CD6, MHC Class II and J5, but it had no effect on the percentages of cells positive for the other T cell markers CD5, CD4, or CD2. Because Con A is a complete mitogen and PDBU is not, the changes observed following Con A stimulation probably reflected an expansion of a particular subpopulation. In contrast, PDBU most likely modifies surface antigen expression directly. Neither treatment affected the B cell subpopulation.

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PubMed1994

Macrophages and Brucella.

The studies reviewed here indicate that during the course of infection with B. abortus protective immune responses occur and that macrophages are activated for antimicrobial activity in both susceptible and resistant animals. Yet in susceptible animals chronic infections with B. abortus become established. There is circumstantial evidence from experiments in vitro that the outcome of infection is related to macrophage/Brucella interactions. This includes the following: 1) the virulence of strains of B. abortus is proportional to their ability to inhibit host phagocyte functions and to survive in murine, bovine, and guinea pig macrophages; and 2) the ability of guinea pig and bovine macrophages to inhibit intracellular growth of brucellae is directly proportional to the innate resistance of the host from which the macrophages were derived. In the murine model, the relative resistance of C57Bl/10 mice to infection with virulent B. abortus strain 2308 correlated with more efficient clearance during the first week following infection, compared with the susceptible BALB/c strain. It is not known whether this was due to innate differences in the recruitment or bactericidal activities of macrophage populations in the two mouse strains, as demonstrated with L. monocytogenes, or to other nonimmune factors such as natural killer cells secreting IFN-gamma and lysing infected macrophages following direct interaction with the brucellae, as shown with other intracellular bacteria. It is also of interest that clearance of strain 2308 following the plateau phase was slower in BALB/c than in C57Bl/10 mice. Since at this time protective T cells are known to be present in the spleens of BALB/c mice, this difference may have been due either to more efficient clearance of brucellae by C57Bl/10 macrophages following activation with T cell cytokines, or to the inability of immune T cells to interact with Brucella-infected macrophages in the susceptible BALB/c strain. CD8 T cells have been shown to have an important role in clearance of brucellae following the peak of infection and may act by lysing chronically infected macrophages. Although protective T cells have been demonstrated in susceptible strains of mice, chronically infected macrophages in these animals may fail to act as targets or may down-regulate T cell functions. As a result, infected macrophages could persist in the presence of Brucella-specific T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1993

Macrophage control of Brucella abortus: role of reactive oxygen intermediates and nitric oxide.

Macrophages infected with Brucella abortus are able to kill intracellular brucellae over the first 12 to 24 hr following infection. Thereafter, the surviving brucellae replicate. We have shown previously that macrophages activated with interferon-gamma (IFN-gamma) have enhanced brucellacidal and brucellastatic activities. In studies reported here, treatment of macrophages with methylene blue, an electron carrier, enhanced the initial killing of intracellular brucellae, indicating their susceptibility to reactive oxygen intermediates. In addition, inhibitors of reactive oxygen intermediates partially blocked the antibrucella activities exhibited by both non-cytokine-treated and IFN-gamma-activated macrophages. In contrast, addition of up to 5 mM NG-monomethyl-L-arginine, to block generation of nitric oxide, resulted in minor but significant levels of blocking of macrophage antibrucella activities only when macrophages were not maximally activated even though maximally activated macrophages produced nitric oxide as indicated by accumulation of nitrite in culture supernatants. In addition, while the J774A.1 macrophage cell line had antibrucella activities which were enhanced by IFN-gamma activation, it did not produce nitric oxide when activated with IFN-gamma and infected with B. abortus. Finally, the IFN-gamma-induced enhancement of antibrucella activities by peritoneal macrophages was inhibited by addition of antitumor necrosis factor-alpha (TNF-alpha) antibodies to the cultures, indicating that TNF-alpha is necessary for full expression of the macrophage antibrucella activities.

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PubMed1993

Iron augments macrophage-mediated killing of Brucella abortus alone and in conjunction with interferon-gamma.

Brucella abortus are Gram negative facultative intracellular bacteria, which survive and replicate in host macrophages. We have recently demonstrated that activation of macrophages with interferon-gamma increases their anti-brucella activities but does not result in elimination of intracellular brucellae. Here we demonstrate that iron-loaded macrophages have an enhanced capacity to kill or prevent replication of intracellular brucellae. Iron added bound to transferrin or as a salt, iron-nitrilotriacetate, can mediate the effect. Macrophages supplemented with iron-loaded transferrin in addition to activation with interferon-gamma can frequently eliminate the intracellular organisms by 48 hr after infection. The effect is apparent following phagocytosis of either nonopsonized or antibody-opsonized brucellae, and with both attenuated and virulent strains of B. abortus. The killing can be blocked by the hydroxyl radical scavengers mannitol and thiourea. This is consistent with the Haber-Weiss reaction, in which iron catalyzes the generation of hydroxyl radicals from hydrogen peroxide.

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PubMed1993

Macrophage control of Brucella abortus: influence of cytokines and iron.

Brucella abortus is a facultative intracellular bacterium that can infect and replicate in mononuclear phagocytes. Recent work has elucidated the role of cytokines in activating macrophages to inhibit the intracellular replication of brucellae, and in recruiting macrophages to the site of infection in vivo. There is also evidence that iron increases the ability of cytokine-activated macrophages to control intracellular brucellae by mechanisms involving reactive oxygen intermediates.

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PubMed1993

Effects of cytokines on intracellular growth of Brucella abortus.

Interleukin 1 alpha (IL-1 alpha), IL-2, IL-4, IL-6, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocyte macrophage colony-stimulating factor (GM-CSF) were tested for their abilities to alter the growth of Brucella abortus in BALB/c J774A.1 murine macrophages. IL-1 alpha, IL-4, IL-6, tumor necrosis factor alpha, and granulocyte macrophage-colony-stimulating factor had no consistent or significant effect on the growth of the avirulent B. abortus strain 19. In contrast, the addition of either IFN-gamma or IL-2 at 100 U/ml to the macrophage cultures resulted in a significant reduction in the number of intracellular bacteria that was not attributable to decreased infection rates. With IL-2, the reduction was most often apparent only during the first 24 h after infection, while inhibition with IFN-gamma was apparent throughout the culture period of 48 h. The addition of either IL-2 or IFN-gamma to macrophage cultures also resulted in reduced intracellular CFU of the virulent B. abortus strain 2308 and the attenuated rough mutant B. abortus strain RB51. Inhibition of intracellular growth was not augmented by combinations of cytokines. Additional studies with IFN-gamma and IL-2 indicated that they could mediate the inhibition of intracellular growth of B. abortus in resident and thioglycolate broth-induced BALB/c peritoneal macrophages and in splenic macrophages. IFN-gamma also inhibited bacterial growth when added after infection of the macrophages, although the magnitude of the antibrucellae effects was less than that when it was added before infection. Furthermore, the maximal inhibitory effect was sustained only when IFN-gamma remained in the cultures after infection of the macrophages.

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PubMed1992

Theileria parva: CD4+ helper and cytotoxic T-cell clones react with a schizont-derived antigen associated with the surface of Theileria parva-infected lymphocytes.

Theileria parva is a protozoan parasite which infects and transforms bovine lymphocytes, resulting in a fatal lymphoproliferative disease. There is evidence that immunity to the intralymphocytic schizont stage is mediated by T cells. We have previously reported derivation of CD4+ T-cell clones which recognize parasite-derived antigens presented on the surface of infected cells in conjunction with MHC molecules and partial characterization of the antigens. The present study further evaluated one of these antigens, demonstrating that it could be derived from cells infected with different parasite stocks as well as from purified theilerial schizonts and that it was recognized by primed, but not unprimed, bovine lymphocytes including cytolytic CD4+ T cells. Using a cloned CD4+ cytolytic cell line, lysis of schizont-infected cells was shown to be MHC-restricted but not parasite-strain restricted. In addition we demonstrated that T cells which respond to the HSS antigen preparation were generated in cattle immunized with parasites from any of the three subspecies of T. parva. The antigenic material was fractionated by sequential subjection to anion-exchange chromatography, hydroxylapatite chromatography, and gel filtration using HPLC, which resulted in recovery of approximately 20% of the antigenic material with more than 10(6)-fold purification in selected fractions. To assess the molecular size of the proteins in the highly purified antigenic fractions, the T. parva-infected lymphocytes were metabolically labeled before fractionation with 3H-amino acids and the material was analyzed by SDS-polyacrylamide gel electrophoresis and liquid scintillation counting of gel slices. The major protein in these fractions had a molecular mass of 9-10 kDa.

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PubMed1992

Immune CD4+ T cells specific for Theileria parva-infected lymphocytes recognize a 24-kilodalton protein.

Theileria parva is a protozoan parasite that infects and transforms bovine lymphocytes. Here we report the partial purification of a T. parva-specific protein from infected lymphocytes that is recognized by CD4+ parasite-specific T-cell clones derived from immune cattle. T. parva-infected lymphocytes were homogenized in Dulbecco's phosphate-buffered saline in the presence of protease inhibitors. The antigen was purified from a postmicrosomal supernatant by using a combination of DEAE-cellulose chromatography and hydroxylapatite column chromatography. After labelling with 125I, the antigen preparation was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to contain 8 to 10 proteins. This preparation was subjected to chromatography in phosphate-buffered saline on HPLC TSK-250/125 columns coupled in tandem. A radiolabelled protein of M(r) 24,000 correlated with antigenic activity.

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PubMed1991

Identification of expressed bovine class I MHC genes at two loci and demonstration of physical linkage.

A cDNA library prepared from lymphocytes of a cow (E98), homozygous at major histocompatibility complex (MHC) loci (BoLA phenotype w10, KN104), was screened with a bovine MHC class I probe. Of the cDNA clones isolated, two, (2.1 and 5.1) were selected and showed divergence at both 5' and 3' termini. E98 DNA was digested with rare-cutter enzymes (Sfi I, Mlu I, Not I, and Cla I) and fragments were size-separated by field inversion gel electrophoresis (FIGE). Hybridization with an entire class I cDNA probe revealed multiple fragments generated by each enzyme. When the 3' untranslated regions (UT) of 2.1 and 5.1 were used as probes, only one fragment was revealed in each digested sample, showing locus specificity of these probes in cattle. Further, DNA of transfected mouse fibroblasts L4 (expressing KN104) and L10 (expressing w10) hybridized to the 3'UT regions of clones 2.1 and 5.1, respectively. Northern blot analysis of the mRNA of the L4 and L10 transfected cells provided further evidence that the cDNA clones 2.1 and 5.1 code for the BoLA-KN104 and BoLA-w10 class I molecules respectively, and thus these represent the products of two different genes. A long range physical mapping of the BoLA-w10 and KN104 genes was performed using FIGE analysis of DNA of an homozygous and an heterozygous animal. This analysis revealed that the BoLA-w10 and KN104 genes are separated by not more than 210 kilobases (kb) and that they are components of a multigene family spanning 1550 kb. As the w10 gene is at the BoLA-A locus we assign the KN104 gene to a B locus.

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PubMed1991

Effects of gamma interferon, tumor necrosis factor alpha, and interleukin-2 on infection and proliferation of Theileria parva-infected bovine lymphoblasts and production of interferon by parasitized cells.

Theileria parva is a protozoan parasite that infects bovine B cells and alpha beta and gamma delta T cells and transforms them into continually proliferating cells. CD4+ T. parva-antigen-specific immune T cells have been shown to produce cytokines in response to stimulation with parasitized cells, and T. parva-infected lymphocytes produce and consume T-cell growth factors and interleukin-2 (IL-2). To ascertain the role of T-cell cytokines on T. parva infections, we evaluated recombinant gamma interferon (rIFN-gamma), rIL-2, and tumor necrosis factor alpha (rTNF-alpha) for their effects on establishment, proliferation, and survival of parasitized cells. The results indicate that neither rIFN-gamma nor rTNF-alpha had an enhancing or inhibitory effect on the growth of established T. parva-infected T-cell clones, whereas bovine rIL-2 increased the proliferation of infected B-cell and alpha beta T-cell clones but not that of gamma delta T-cell clones. To evaluate the effects of the cytokines on establishment of parasitized cell lines, peripheral blood mononuclear cells were cultured in their presence immediately following infection with T. parva sporozoites. Neither rIFN-gamma nor rIL-2 altered the proportion of cells initially developing schizonts, but both enhanced establishment of infected cell lines by about twofold. In contrast, rTNF-alpha resulted in about a 33% decrease in the proportion of schizont-infected cells. Inhibitory effects on establishment of parasitized cell lines by rTNF-alpha were no longer apparent by 12 days following infection. Tests conducted during this study indicated that T. parva-infected lymphocytes also spontaneously produce IFN that is neutralized by acidic pH treatment. In conclusion, we speculate that none of these T-cell cytokines are likely to have a profound inhibitory effect in vivo on T. parva infections. Instead, IFN-gamma and IL-2 may facilitate the establishment of infection by T. parva.

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PubMed1990

Identification of a bovine surface antigen uniquely expressed on CD4-CD8- T cell receptor gamma/delta+ T lymphocytes.

In this study, two monoclonal antibodies, IL-A29 and CC15, are described that identify a novel bovine cell surface marker of 215/300 kDa. The antibodies reacted with a discrete population of resting lymphocytes in peripheral blood which, in young animals, constituted about 25% of the mononuclear cells. Thymus, lymph nodes and spleen contained less than 5% positive cells. These cells were negative for surface Ig, a monocyte/granulocyte marker, and the T lymphocyte antigens CD2, CD6, CD4 and CD8. Immunohistological analyses revealed the presence of IL-A29/CC15-positive lymphocytes in the thymic medulla, in the outer cortex of lymph nodes, in the marginal zones of the spleen, in the dermal and epidermal layers of the skin and in the lamina propria of the gut. The IL-A29/CC15+ cells in unfractionated blood mononuclear cells responded in autologous and allogeneic mixed lymphocyte cultures, and when purified they responded to concanavalin A in the presence of recombinant interleukin 2. These observations suggested this population of cells belonged to the T cell lineage. In order to unambiguously define their lineage, cDNA clones encoding bovine T cell receptor (TcR) and CD3 proteins were isolated. Northern blot analyses of IL-A29/CC15+ cell populations and of established cell lines of various lineages demonstrated that they expressed TcR delta and CD3 gamma, delta and epsilon mRNA: TcR alpha was not expressed, whereas only a truncated form of TcR beta mRNA was present. These results indicate that the IL-A29 and CC15 antibodies define a unique population of CD4-CD8-, gamma/delta T cells.

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PubMed1990

Immunization with Theileria parva parasites from buffaloes results in generation of cytotoxic T cells which recognize antigens common among cells infected with stocks of T. parva parva, T. parva bovis, and T. parva lawrencei.

Immunity to infection by the protozoan parasite Theileria parva in cattle is partially attributable to cytotoxic T cells, which kill lymphocytes infected with the schizont stage of the parasite. Here we evaluated five stocks of buffalo-derived T. parva lawrencei parasites and two stocks of cattle-derived T. parva parva parasites for their ability to induce in vivo cytotoxic T cells which can kill lymphocytes infected with a wide variety of strains of T. parva parasites. A group of seven full-sibling cattle, produced by embryo transfer and matched for at least one major histocompatibility complex class I haplotype, were immunized by infection and treatment with the parasite stocks. Target cells used in in vitro cytotoxicity assays were infected with five buffalo-derived parasite stocks and five cattle-derived parasite stocks, including T. parva parva and T. parva bovis. Immunization with any of the seven parasite stocks resulted in the generation of cytotoxic T cells which recognized parasite antigens on most if not all of the target cell lines tested, although the T. parva bovis stock was the least effective at doing so. Further in-depth analyses performed with peripheral blood mononuclear cells from one of the cattle immunized with T. parva lawrencei parasites showed that the pattern of killing of the panel of target cells was altered when either cells infected with different parasite stocks or clones of infected cells were used as stimulator cells in vitro, suggesting the presence of more than one population of parasite-specific cytotoxic effector cells in the peripheral blood mononuclear cells. However, clones of these cytotoxic effector cells recognized common or cross-reactive antigen epitopes expressed by the entire panel of infected target cells. These T-cell clones will be useful for identifying common T-cell antigen epitopes of T. parva and the parasite genes encoding them.

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PubMed1990

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed.

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PubMed1989

Infection of bovine T cell clones with genotypically distinct Theileria parva parasites and analysis of their cell surface phenotype.

Different stocks and stabilates within a stock of Theileria parva were analysed for genotypic differences and for their effect on the expression of host cell surface antigens following infection of BoT8+ T lymphocyte clones. The parasites were characterized in vitro by hybridization of T. parva-specific DNA probes to Southern blots of endonuclease-digested DNA from the infected T cell clones. Phenotypic changes in the host lymphoblastoid cells before and after infection were examined using lineage-specific monoclonal antibodies which reacted with the differentiation antigens BoT2, BoT4, Bo6, BoT8 and a null cell marker on bovine T cells. Expression of Class I and Class II major histocompatibility complex (MHC) antigens on the cell populations was also assessed. Results of this study indicate that genotypically different parasites exist among and within T. parva stabilates and that the expression of Bo6, BoT8 and the null cell marker was differentially altered by infection with parasites from different stocks or from different stabilates of the same stock. Expression of Class II antigens was significantly increased after infection. Moreover, clones that were derived from the same cell line but had genotypically distinct T. parva parasites, also showed differences in expression of Bo6 and BoT8 and the null cell marker.

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