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Biomedical subjects

R M Locksley

Publications and source records attributed to R M Locksley.

At least 55 records · Page 3Linked to original sources
PubMed1996

Beta 2-microglobulin-dependent NK1.1+ T cells are not essential for T helper cell 2 immune responses.

A number of investigations have established the critical role of interleukin 4 (IL-4) in mediating the development of T helper (Th)2 effector cells in vitro and in vivo. Despite intensive study, the origin of the IL-4 required for Th2 priming and differentiation remains unclear. Natural killer (NK)1.1+ alpha/beta T cell receptor+ T(NT) cells, a unique lineage of cells capable of producing large amounts of IL-4 after activation in vivo, are important candidates for directing Th2 priming. These cells are selected by the nonpolymorphic major histocompatibility complex (MHC) class I molecule, CD1, and are deficient in beta 2-microglobulin (beta 2m)-null mice. We used beta 2m-deficient mice on both BALB/c and C57BL/6 backgrounds to examine their capacity to mount Th2 immune responses after challenge with a number of well-characterized antigens administered by a variety of routes. As assessed by immunization with protein antigen, infection with Leishmania major, embolization with eggs of Schistosoma mansoni, intestinal infection with Nippostrongylus brasiliensis, or induction of airway hyperreactivity to aerosolized antigen, beta 2m-deficient mice developed functional type 2 immune responses that were not substantially different than those in wild-type mice. Production of IL-4 and the generation of immunoglobulin E (IgE) and eosinophil responses were preserved as assessed by a variety of assays. Collectively, these results present a comprehensive analysis of type 2 immune responses in beta 2m-deficient mice, and indicate that beta 2m-dependent NT cells are not required for Th2 development in vivo.

Animals↗
PubMed1996

Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10.

The mechanism by which the mammalian mother accepts the implanting fetus as an allograft remains unexplained, but is likely to be the result of a combination of factors. Mononuclear cytotrophoblasts, the specialized fetal cells of the placenta that invade the uterus, play an important role. These cells express HLA-G, an unusual major histocompatibility complex class I-B molecule, and secrete cytokines and pregnancy-specific proteins that can regulate immune function. We investigated whether cytotrophoblasts secrete interleukin 10 (IL-10), a cytokine that potently inhibits alloresponses in mixed lymphocyte reactions. Cytotrophoblasts from all stages of pregnancy produced IL-10 in vitro, but neither placental fibroblasts nor choriocarcinoma (malignant trophoblast) cell lines did so. Spontaneous IL-10 production averaged 650, 853, and 992 pg/10(6) cells in the first, second, and third trimesters of pregnancy, respectively. IL-10 secretion dropped approximately 10-fold after the first 24 h of culture, and was paralleled by a decrease in messenger RNA. IL-10 messenger RNA was detected in biopsies of the placenta and the portion of the uterus that contains invasive cytotrophoblasts, suggesting that this cytokine is also produced in vivo. IL-10 secreted by cytotrophoblasts in vitro is bioactive, as determined by its ability to suppress interferon gamma production in an allogeneic mixed lymphocyte reaction. We conclude that human cytotrophoblast IL-10 may be an important factor that contributes to maternal tolerance of the allogeneic fetus.

Base Sequence↗
PubMed1996

The instructive role of innate immunity in the acquired immune response.

Innate immunity has been considered only to provide rapid, incomplete antimicrobial host defense until the slower, more definitive acquired immune response develops. However, innate immunity may have an additional role in determining which antigens the acquired immune system responds to and the nature of that response. Knowledge of the molecules and pathways involved may create new therapeutic options for infectious and autoimmune diseases.

Animals↗
PubMed1996

Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway hyperreactivity.

Reversible airway hyperreactivity underlies the pathophysiology of asthma, yet the precise mediators of the response remain unclear. Human studies have correlated aberrant activation of T helper (Th) 2-like effector systems in the airways with disease. A murine model of airway hyperreactivity in response to acetylcholine was established using mice immunized with ovalbumin and challenged with aerosolized antigen. No airway hyperractivity occurred in severe combined immunodeficient mice. Identically immunized BALB/c mice developed an influx of cells, with a predominance of eosinophils and CD4+ T cells, into the lungs and bronchoalveolar lavage fluid at the time that substantial changes in airway pressure and resistance were quantitated. Challenged animals developed marked increases in Th2 cytokine production, eosinophil influx, and serum immunoglobulin E levels. Neutralization of interleukin (IL) 4 using monoclonal antibodies administered during the period of systemic immunization abrogated airway hyperractivity but had little effect on the influx of eosinophils. Administration of anti-IL-4 only during the period of the aerosol challenge did not affect the subsequent response to acetylcholine. Finally, administration of anti-IL-5 antibodies at levels that suppressed eosinophils to < 1% of recruited cells had no effect on the subsequent airway responses. BALB/c mice had significantly greater airway responses than C57BL/6 mice, consistent with enhanced IL-4 responses to antigen in BALB/c mice. Taken together, these data implicate IL-4 generated during the period of lymphocyte priming with antigen in establishing the cascade of responses required to generate airway hyperractivity to inhaled antigen. No role for IL-5 or eosinophils could be demonstrated.

Acetylcholine↗
PubMed1996

Leishmania major: targeting IL-4 in successful immunomodulation of murine infection.

Protection against Leishmania major infection among inbred strains of mice is dependent upon successful expansion and activation of type 1 CD4+ effector (Th1) cells, a process that is aberrant in highly susceptible BALB strains. We sought to establish whether vaccination strategies using whole parasite lysates or a characterized immunodominant antigen, the Leishmania homolog of mammalian receptor for activated protein kinase C (LACK), would be capable of protecting subsequently infected BALB mice if given within a cytokine milieu capable of biasing the immune response toward Th1 cells. When given with neutralizing antibody to IL-4, but not when given alone, subcutaneously administered soluble Leishmania antigens mediated substantial protection to BALB/c mice against subsequent infection with parasites as assessed by size of the local lesion, enhanced Th1-type immune responses, and decreased parasite burdens. Similarly, when given with recombinant IL-12, LACK conferred substantial protection to cohorts of BALB.B, BALB/c, and BALB.K mice that was associated with reduction in serum IgE levels, consistent with effects on IL-4 production. Thus altering the cytokine milieu during administration of vaccine antigens by neutralizing IL-4 induced powerful Th1 recall responses during infection that were capable of mediating substantial levels of protection.

Animals↗
PubMed1996

Cytokine production by peripheral blood mononuclear cells in human coccidioidomycosis.

The production and mRNA expression of the cytokines interferon-gamma (IFN-gamma) and interleukin (IL)-4, -10, and -12 by peripheral blood mononuclear cells (PBMC) after incubation with the coccidioidal antigen toluene spherule lysate (TSL) from various subjects were measured. The IFN-gamma concentration in PBMC supernatants incubated for 72 h from 8 subjects with disseminated coccidioidomycosis was significantly less than that from 7 healthy, coccidioidal-immune subjects (P = .015). No differences were seen among the subject groups in the concentrations of IL-4, -10, or -12. By use of competitive polymerase chain reaction, PBMC from subjects with disseminated coccidioidomycosis also expressed less mRNA for IFN-gamma and IL-12 than did cells from healthy, immune subjects. These data suggest that patients with disseminated coccidioidomycosis have a diminished T helper lymphocyte type 1 response.

Base Sequence↗
PubMed1995

Expression cloning of a protective Leishmania antigen.

Parasite-specific CD4+ T cells have been shown to transfer protection against Leishmania major in susceptible BALB/c mice. An epitope-tagged expression library was used to identify the antigen recognized by a protective CD4+ T cell clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histocompatibility complex class II. A conserved 36-kilodalton member of the tryptophan-aspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12 before infection.

Amino Acid Sequence↗
PubMed1995

The development of effector T cell subsets in murine Leishmania major infection.

Leishmania major infection has proven an exceptional model for CD4+ subset development in inbred mice. Most strains contain infection coincident with the appearance of T helper 1 (Th1) cells that produce gamma-interferon (IFN-gamma) required for macrophage activation. In contrast, mice on the BALB background are unable to control infection due to the development of Th2 cells that produce counter-regulatory cytokines, particularly interleukin 4 (IL-4), capable of abrogating the effects of IFN-gamma. Selective gene disruption studies in mice have illustrated critical components of the host response to L. major. Mice deficient in beta 2 microglobulin, which have no major histocompatibility complex (MHC) class I or CD8+ T cells, control infection as well as wild-type mice, whereas mice deficient in MHC class II (and CD4+ T cells) suffer fatal infection. Mice with disruption of the gene coding IFN-gamma are also incapable of containing infection, reflecting absolute requirements for this cytokine. A number of interventions have been demonstrated to abrogate Th2 cell development in BALB mice, enabling these mice to control infection. Each of these--IL-12, anti-IL-4, anti-IL-2, anti-CD4 and CTLA4-Ig--has in common the capacity to make IL-4 rate limiting at the time of CD4+ cell priming.

Animals↗
PubMed1995

The regulation of immunity to Leishmania major.

Experimental infection with the intracellular protozoan Leishmania major constitutes a particularly versatile model for assessing the role of CD4+ subset development in the host response to infectious disease. The association of Th1 development with control of infection, and of Th2 cell development with progressive disease, has been well established. The capacity to manipulate the outcome, using distinct immunologic interventions, in both genetically resistant and susceptible mice has identified key effector cytokines that must be present during the time of initial priming of T cells in order to affect the CD4 switch phenotype. Roles for interferon-gamma (IFN-gamma), interleukin 12 (IL-12), and IL-4 in Th1 and Th2 maturation have been demonstrated, although additional undefined signals are required. Study of the genetically susceptible BALB/c mouse has shown failure to downmodulate IL-4 production in response to infection, a response that is critically associated with the failure to develop appropriate Th1 responses. Use of the murine L. major model continues to elucidate new methods for vaccine development and suggests a promising system for identification of genes that determine susceptibility to infection.

Animals↗
PubMed1994

Interferon gamma-independent effects of interleukin 12 administered during acute or established infection due to Leishmania major.

Interleukin 12 (IL-12) is a powerful stimulus for the growth of activated T and natural killer cells, their generation of interferon gamma (IFN-gamma), and the differentiation of T helper type 1 (Th1) effector cells from naive precursors in vitro. These activities are consistent with the capacity of exogenous IL-12 to heal otherwise susceptible BALB/c mice infected with the intramacrophage parasite Leishmania major. Using this characterized model of CD4 cell subset differentiation, we examined the immunologic effects of IL-12 administered either at the time of infection, when naive T cells are primed, or after 14 days of infection, by which time CD4+ subset differentiation has occurred. Given with the inoculation of parasites, IL-12 induced IFN-gamma and IL-10 and markedly suppressed IL-4. Effects on IL-10 and IL-4 were comparable in mice with homozygous disruption of the IFN-gamma gene (IFN-gamma 0/0), and suppression of IL-4 was unchanged by administration of neutralizing anti-IL-10 antibody. Induction of IFN-gamma and IL-10 mRNA by IL-12 also occurred in infected SCID mice. Given after day 14 of infection, however, IL-12 not only induced IFN-gamma and IL-10 but also induced IL-4 in normal and IFN-gamma 0/0 mice. These data demonstrate direct effects of IL-12 independent of IFN-gamma, IL-10, and IL-4 and demonstrate that the ineffectiveness of IL-12 administered following infection with L. major correlates with resistance of differentiated Th2 cells to the IL-4-suppressing activity of IL-12.

Animals↗
PubMed1994

Differential effects of blockade of CD28-B7 on the development of Th1 or Th2 effector cells in experimental leishmaniasis.

Infection of inbred strains of mice with Leishmania major is a well-characterized model for analysis of the development of effector CD4+ subsets of the Th1 and Th2 types in vivo. We co-administered a fusion protein, CTLA4Ig, that blocks the CD28-B7 costimulatory pathway important for optimal T cell activation, to assess the relative role for this pathway during maturation of Th1 and Th2 cells in vivo. Surprisingly, CTLA4Ig administered within the first week of infection completely abrogated progressive disease in susceptible BALB/c mice while having no effect on the protective immune response developed by resistant C57BL/6 mice. The protective effect in BALB/c mice was increasingly lost if administration of CTLA4Ig was delayed longer than 1 wk after infection. As in other protective interventions used in this model, control of infection was associated with down-regulation of IL-4 mRNA transcripts in lymph node cells recovered 5 wk after infection together with abrogation of IgE production and enhanced parasite-specific IgG2a relative to IgG1. Although a single dose of CTLA4Ig was protective, sustained delivery abolished the capacity of BALB/c mice to contain infection, suggesting that costimulation through this pathway is required at later stages of the immune response. Taken together, the data demonstrate that the priming of Th2 cells is more dependent upon the CD28-B7 pathway than the priming of Th1 cells, and suggest that the development of Th subsets in vivo may be influenced by limiting CD28-B7 costimulation.

Abatacept↗
PubMed1994

CD4+ effector cells default to the Th2 pathway in interferon gamma-deficient mice infected with Leishmania major.

Mice with homologous disruption of the interferon gamma (IFN-gamma) gene on the C57BL/6 background were infected with Leishmania major and the immune response assessed. In contrast to wild-type or heterozygous knockout mice, deficient animals were unable to restrict growth of the parasite and suffered lethal infection over 6-8 wk. Although wild-type and heterozygous littermates developed CD4+ cells that contained transcripts for IFN-gamma and lymphotoxin, typical of T helper type 1 (Th1) cells, the knockout mice developed CD4+ cells that contained transcripts for interleukin 4 (IL-4), IL-5, and IL-13, typical of Th2 cells. ELISPOT assays confirmed the reciprocal patterns of IFN-gamma or IL-4 production by T cells in similar frequencies in the respective groups of mice, and antibody analysis confirmed the presence of Th2-mediated isotype switching in the knockout mice. These data suggest that CD4+ T cells that normally respond to antigens by differentiation to Th1 cells default to the Th2 pathway in the absence of endogenous IFN-gamma.

Animals↗
PubMed1994

Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection.

Leishmania major are intramacrophage parasites whose eradication requires the induction of T helper 1 (Th1) effector cells capable of activating macrophages to a microbicidal state. Interleukin 12 (IL-12) has been recently identified as a macrophage-derived cytokine capable of mediating Th1 effector cell development, and of markedly enhancing interferon gamma (IFN-gamma) production by T cells and natural killer cells. Infection of macrophages in vitro by promastigotes of L. major caused no induction of IL-12 p40 transcripts, whereas stimulation using heat-killed Listeria or bacterial lipopolysaccharide induced readily detectable IL-12 mRNA. Using a competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed. All strains of mice examined, including susceptible BALB/c and resistant C57BL/6, B10.D2, and C3H/HeN, had the appearance of a CD4+ population in the draining lymph nodes that contained transcripts for IL-2, IL-4, and IFN-gamma (and in some cases, IL-10) that peaked 4 d after infection. In resistant mice, the transcripts for IL-2, IL-4, and IL-10 were subsequently downregulated, whereas in susceptible BALB/c mice, these transcripts were only slightly decreased, and IL-4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection, consistent with induction by intracellular amastigotes. Challenge of macrophages in vitro confirmed that amastigotes, in contrast to promastigotes, induced IL-12 p40 mRNA. Reexamination of the cytokine mRNA at 4 d revealed expression of IL-13 in all strains analyzed, suggesting that IL-2 and IL-13 may mediate the IL-12-independent production of IFN-gamma during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in CD4+ T cells. Each of these activities may favor survival of the organism.

Animals↗
PubMed1993

Constructing polycompetitor cDNAs for quantitative PCR.

Analysis of mRNA levels using reverse transcription coupled with the polymerase chain reaction provides a powerful tool for studying cytokine regulation in cellular immunology. We report a novel method for cloning competitor cDNAs that is rapid, efficient and inexpensive. By linking multiple competitor cDNAs in tandem, polycompetitor constructs can be created that allow the use of a single reagent for individual PCR assays. Assays can be performed on minute samples of cell culture or tissue and can be reliably quantitated after routine gel electrophoresis without the use of densitometry or labeled nucleotides. The utility of this technique lies in the ability to produce a relatively inexpensive customized reagent that is simple to use and that allows for sensitive determinations of gene expression in a rapid and convenient manner. This method should allow investigators in many areas of biology to easily quantitate a broad range of important regulatory molecules.

Animals↗