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R M Locksley

Publications and source records attributed to R M Locksley.

At least 73 records · Page 4Linked to original sources
PubMed1993

Helper T cells without CD4: control of leishmaniasis in CD4-deficient mice.

Expression of either the CD4 or CD8 glycoproteins discriminates two functionally distinct lineages of T lymphocytes. A null mutation in the gene encoding CD4 impairs the development of the helper cell lineage that is normally defined by CD4 expression. Infection of CD4-null mice with Leishmania has revealed a population of functional helper T cells that develops despite the absence of CD4. These CD8- alpha beta T cell receptor+ T cells are major histocompatibility complex class II-restricted and produce interferon-gamma when challenged with parasite antigens. These results indicate that T lymphocyte lineage commitment and peripheral function need not depend on the function of CD4.

Animals↗
PubMed1993

Targeted activation of CD8 cells and infection of beta 2-microglobulin-deficient mice fail to confirm a primary protective role for CD8 cells in experimental leishmaniasis.

CD8+ T cells play an important role in the immunologic control of intracellular pathogens, particularly viruses. Leishmania are obligate intracellular parasites of macrophages in the mammalian host, and previous studies using deletion of CD8+ cells by administration of mAb to infected animals have suggested a protective role for these cells. Two complementary approaches were used to define more carefully the role of CD8+ cells in leishmaniasis. In BALB/c mice susceptible to Leishmania major (L. major) infection, targeted activation of CD8+ T cells was attempted by immunization with nonapeptides derived from the conserved major outer surface protein of the organism, gp63, that contained the consensus binding motif for MHC class I H-2Kd molecules. Two of the nonapeptides induced CTL activity in subsequently infected BALB/c mice that could be elicited against P815 cells pulsed either with peptide or lysates of L. major. Purified CD8+ T cells from immunized mice had elevated levels of IFN-gamma mRNA transcripts as compared to unimmunized mice. Despite evidence for activation of CD8+ cells, none of the mice immunized with nine different peptides alone or in combination were protected from progressive disease. In a second series of experiments, beta 2-microglobulin deficient mice that lack CD8+ cells were infected with L. major and the course of infection monitored. These mice cured disease as rapidly as beta 2-m +/- and +/+ littermates, and cure was associated with comparable levels of IFN-gamma mRNA in the draining lymph node population. Neither of these approaches was able to confirm a substantive role for CD8+ T cells in the primary protective response to L. major.

Amino Acid Sequence↗
PubMed1993

The 31-kDa precursor of interleukin 1 alpha is myristoylated on specific lysines within the 16-kDa N-terminal propiece.

The cytokine interleukin 1 alpha (IL-1 alpha) is a critical mediator of the immune and inflammatory responses. A unique determinant of its activity as compared with IL-1 beta may be its association with the plasma membrane. While the biologic activity of "membrane IL-1" has been extensively reported, the mechanism of membrane binding remains unclear. We report that the N terminus of the 31-kDa IL-1 alpha precursor is myristoylated on specific internal lysine residues. Immunoprecipitation of [3H]myristic acid-radiolabeled human monocyte lysates with IgG antibodies to the 31-kDa IL-1 alpha precursor recovered a protein with the physicochemical properties of the IL-1 alpha N-terminal propiece (16 kDa, pI 4.45). Glycyl N-myristoylation of this protein is precluded by the absence of a glycine residue at position 2, suggesting that the propiece is myristoylated on epsilon-amino groups of lysine. To determine which lysine(s) are acylated, a series of synthetic peptides containing all lysines found in the IL-1 alpha N-terminal propiece were used in an in vitro myristoylation assay containing peptide, myristoyl-CoA, and monocyte lysate as enzyme source. Analysis of the reaction products by reverse-phase HPLC and gas-phase sequencing demonstrated the specific myristoylation of Lys-82 and Lys-83, yielding predominantly monoacylated product. A conserved sequence in the IL-1 beta propiece was myristoylated with at least 8-fold less efficiency. Acylation of the IL-1 alpha precursor by a previously unrecognized lysyl epsilon-amino N-myristoyl-transferase activity may facilitate its specific membrane targeting.

Acylation↗
PubMed1993

Transfected Leishmania expressing biologically active IFN-gamma.

Infection of susceptible BALB/c mice with Leishmania major leads to progressive infection with the failure to expand and activate Th1 CD4+ T cells that elaborate IFN-gamma, a critically implicated cytokine for control of disease. We used the recently described capacity to express foreign genes in trypanosomatids to introduce into Leishmania the murine IFN-gamma gene on a drug-selectable plasmid under the constitutive control of intergenic tubulin sequences. Several clones of L. major were established and demonstrated to contain IFN-gamma DNA and IFN-gamma RNA that was appropriately trans-spliced with the Leishmania-specific leader sequence, and to secrete IFN-gamma into the media. The secreted IFN-gamma was biologically active as assessed by up-regulation of class II MHC Ag and induction of macrophage nitric oxide synthase activity in a macrophage cell line. Infection of nude mice with IFN-gamma-containing organisms resulted in significantly slower progression of disease as compared to infection with organisms containing the empty plasmid, suggesting that biologically important activation of infected macrophages might be occurring in vivo. Infection of genetically susceptible BALB/c mice, however, did not impede the expansion of Th2 cells and the inexorable progression of disease. Despite the demonstration of increased levels of IFN-gamma transcription in vivo, induction of nitric oxide synthase in macrophages and expression of Ly-6, and IFN-gamma-inducible Ag, on CD4+ lymphocytes could not be shown. In all cases, organisms recovered from tissue amastigotes contained the IFN-gamma plasmid and secreted active IFN-gamma. The data confirm earlier studies that IFN-gamma alone is not sufficient to impede activation and maturation of Th2 cells in susceptible mice, even when targeted directly to the infected cell.

Animals↗
PubMed1993

IL-2 is necessary for the progression of leishmaniasis in susceptible murine hosts.

BALB/c mice are highly susceptible to disseminated infection with the intracellular protozoa Leishmania major. Progression of disease requires in vivo expansion of Th2 CD4+ lymphocytes and is reversed by treatment with anti-IL-4 monoclonal antibody. Inasmuch as IL-2 may be necessary for both the production of IL-4 and differentiation of Th2 cells, the possible contribution of IL-2 to progressive infection was examined. Four weekly injections of anti-IL-2 mAb (S4B6) cured more than 80% of BALB/c mice infected with L. major, as determined by diminished footpad swelling and decreased numbers of parasites in infected tissues. Multiple doses of S4B6 were necessary for benefit; a single dose given at the time of infection was ineffective. The anti-IL-2R mAb PC61 demonstrated a similar protective effect when administered twice weekly for 4 wk. Anti-IL-2-mediated cure of cutaneous leishmaniasis was associated with increased IFN-gamma and decreased IL-4 production by regional lymph node cells compared to untreated BALB/c mice with progressive illness. Both CD4+ and CD8+ T lymphocytes contributed to the increased expression of IFN-gamma mRNA in cured mice. These data suggest that levels of IL-2 suboptimal for Th2 expansion in vivo do not inhibit Th1 CD4+ and CD8+ T cell activation and IFN-gamma synthesis. Other cytokines or activation pathways that are either IL-2-independent or synergistic with low levels of IL-2 may account for the appearance of curative T cell responses during treatment with anti-IL-2 antibodies.

Animals↗
PubMed1993

TH1 and TH2 cell antigen receptors in experimental leishmaniasis.

The complexity and chronicity of parasitic infections have obscured the identification of biologically relevant antigens. Analysis of the T cell receptor repertoire used by mice infected with Leishmania major revealed the expansion of a restricted population of CD4+ cells. These cells expressed the V alpha 8-J alpha TA72, V beta 4 heterodimer in both progressive infection and protective immunity and across several major histocompatibility haplotypes. Thus, the same immunodominant parasite epitope drives the disparate outcomes of this infectious process, suggesting that candidate vaccine antigens selected by screening of immune individuals may be capable of exacerbating disease in genetically susceptible individuals.

Animals↗
PubMed1993

Cytokines in the differentiation of Th1/Th2 CD4+ subsets in leishmaniasis.

Leishmania major infect only macrophages in the host, where they reside in endolysosomal compartments into which MHC class II molecules co-localize. Experimental infection in mice has provided a useful model for the differentiation of Th1 CD4+ effector lymphocytes that are required for the generation of IFN-gamma that activates the macrophage to a microbicidal state. Genetically susceptible BALB/c mice aberrantly activate Th2 CD4+ effector cells that are ineffective in arresting infection. Increasing evidence suggests that, rather than discrete parasite antigens or MHC molecules, cytokines mediate the critical decision in the developmental switch to either the Th1 or Th2 effector phenotype.

Animals↗
PubMed1993

The worm and the protozoa: stereotyped responses or distinct antigens?

Studies of parasitic diseases have provided the best in vivo correlates of the division of CD4+ helper T cells into distinct functional phenotypes, designated T(H)I and T(H)2, that mediate the balanced regulation of cellular and humoral immunity. In this article, Steven Reiner and Richard Locksley focus on why parasitic infections tend to generate such clearly polarized responses and emphasize that early events that mediate maturation signals towards T(H)1- or T(H)2-effector and memory cells remain incompletely defined. Effective vaccination that seeks to mold the developing immune response will need to consider the role of interleukins and various cell-surface molecules that have been identified, thus far, to influence CD4 subset differentiation.

Journal Article↗
PubMed1993

Potential role for interleukin-10 in the immunosuppression associated with kala azar.

Patients with acute kala azar are generally nonreactive in a number of immunologic assays, including T cell proliferation and generation of macrophage-activating cytokines, principally IFN-gamma, in response to leishmania antigens in vitro. To test for potential immunosuppressive factors, a series of T cell lines and clones were established from patients with acute kala azar, from patients after chemotherapy for kala azar, and from skin test-positive adults from the same endemic region. Although CD4+ T cell lines and clones could be readily established from the skin test-positive adults, lines and clones from acute or treated patients were heavily biased in expression of CD8+. The CD8+ cells from acute patients did not themselves release cytokines in response to leishmania antigens in vitro, but markedly affected the cytokine profile of peripheral blood mononuclear cells isolated 1 yr later after recovery. Addition of the CD8+ cells caused inhibition of lymphoproliferation and IFN-gamma release, with augmentation of IL-6 and IL-10 release. The inhibitory effects of the CD8+ cells could be partially abrogated by antibodies to IL-10 but not by antibodies to IL-4. Analysis of four patients with acute kala azar demonstrated release of IL-10 that could not be demonstrated in supernatants from asymptomatic skin test-positive individuals. Generation of IL-10 may contribute to the profound suppression of IFN-gamma release that occurs during kala azar due to Leishmania chagasi.

Adolescent↗
PubMed1993

Reconstitution of Mycobacterium leprae immunity in severe combined immunodeficient mice using a T-cell line.

To test whether Mycobacterium leprae-immune T cells can confer protection against infection with leprosy bacilli, severe combined immunodeficient (SCID) mice were reconstituted with a BALB/c-derived, M. leprae-responsive, T-cell line. Flow cytometric analysis of spleen and peripheral blood cells confirmed reconstitution with T cells. In vitro lymphokine production and the proliferation of spleen cells from the reconstituted animals established that the donor cells had maintained their functional activity for the duration of the study (275 days). The transfer of immune T cells 24 hr before foot pad infection with leprosy bacilli resulted in a profound reduction in M. leprae multiplication, as compared to the nonreconstituted SCID mice. The yield of acid-fast bacilli in the foot pads of SCID mice reconstituted with the M. leprae-immune T cells also was significantly lower than that found in naive BALB/c mice, and at levels previously found only in BALB/c mice that had been immunized effectively. These experiments demonstrate that M. leprae-immune T cells home effectively and control M. leprae infection in SCID mice.

Animals↗
PubMed1992

Myristyl acylation of the tumor necrosis factor alpha precursor on specific lysine residues.

NH2-terminal glycine myristyl acylation is a cotranslational modification that affects both protein localization and function. However, several proteins that lack NH2-terminal glycine residues, including the interleukin 1 (IL-1) precursors, also contain covalently linked myristate. To date, the site(s) of acylation of these proteins has not been determined. During an evaluation of IL-1 acylation, it was observed that [3H]myristate-labeled human monocyte lysates contained a prominent 26-kD myristylated protein, which was identified as the tumor necrosis factor alpha (TNF) precursor protein on the basis of specific immune precipitation. Radioimmunoprecipitates from the supernates of labeled monocytes indicated that the processed or mature 17-kD form of TNF does not contain myristate, suggesting that the site of acylation occurs within the 76-amino acid propiece of the precursor molecule. As the TNF precursor does not contain an NH2-terminal glycine, we hypothesized that myristyl acylation occurs on the N-epsilon-NH2 groups of lysine, of which two are present in the propiece (K19K20). Synthetic peptides were designed to include all seven lysine residues present within the entire 26-kD TNF precursor, and used in an in vitro myristyl acylation assay containing peptide, myristyl-CoA, and monocyte lysate as a source of enzyme. Analysis of reaction products by reverse phase high performance liquid chromatography and gas phase sequencing demonstrated the exclusive myristyl acylation of K19 and K20, consistent with the presence in monocytes of a specific lysyl N-epsilon-NH2-myristyl transferase activity. The acylated lysine residues are located immediately downstream from a hydrophobic, probable membrane-spanning segment of the propiece. Specific myristyl acylation of the TNF propiece may facilitate membrane insertion or anchoring of this critical inflammatory mediator.

Acylation↗
PubMed1992

Tumour necrosis factor alpha restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice.

Schistosomiasis (bilharzia) is a parasitic disease caused by several species of schistosome worms (blood flukes). The key pathogenic event in this disease is the formation of granulomas around schistosome eggs trapped in portal venules of the liver. Granulomas are a distinctive form of chronic inflammation characterized by localized aggregation of activated macrophages around an inciting stimulus. Each granuloma evolves to form a fibrous scar; in schistosomiasis, the result is widespread hepatic fibrosis and portal hypertension. To identify the specific immune signal molecules necessary for granuloma formation, we studied schistosome infections in severe combined immunodeficient (SCID) mice, which have normal macrophages but lack functional B or T lymphocytes. Here we report that the immunoregulatory cytokine tumour necrosis factor alpha is necessary and sufficient to reconstitute granuloma formation in schistosome-infected SCID mice. Moreover, we find that the parasitic worms require tumour necrosis factor alpha for egg-laying and for excretion of eggs from the host. The implication of this latter result is that the parasite has adapted so successfully to its host that it uses a host-derived immunoregulatory protein as a signal for replication and transmission.

Animals↗
PubMed1992

Interleukin 1: the patterns of translation and intracellular distribution support alternative secretory mechanisms.

Interleukin-1 (IL-1) is synthesized as a 31 kDa precursor protein, whose multiple extracellular activities are attributed to receptor binding of a processed, carboxy-terminal 17 kDa peptide. Unlike other secreted proteins, the IL-1 precursor lacks a hydrophobic leader sequence and is not found in organelles composing the classical secretory pathway. In order to further clarify the intracellular processing of IL-1, we studied its site of synthesis in human monocytes. Secreted and integral membrane proteins are translated on membrane-bound polyribosomes, while intracellular proteins are translated on free polyribosomes. Free and membrane-bound polysomes were isolated from Lipid A-stimulated monocyte lysates and immunoblotted using antibodies specific to the N-terminal regions of the IL-1 alpha and beta precursors. Free polysome fractions showed multiple small bands consistent with nascent peptide chains; membrane-bound polysomes yielded no detectable IL-1. Polysome fractions were then analyzed by immunoelectron microscopy; nascent IL-1 alpha and beta peptide chains were readily seen emerging from cytoskeletal-associated free polyribosomes, but not membrane-bound polyribosomes. Electron microscopic in situ hybridization revealed IL-1 mRNA chains attached to cytoskeletal-associated free, but not membrane-bound polyribosomes. The intracellular distribution of the fully synthesized IL-1 beta precursor was studied in human mesangial cells (HMC), whose cytoskeletal organization is more readily evaluated than that of monocytes. Dual immunofluorescence microscopy of these cells revealed a complex intracellular distribution of the fully synthesized 31 kDa IL-1 precursors. IL-1 was asymmetrically distributed between cytosolic, microtubule, and nuclear compartments, without association with actin or intermediate filaments. This demonstration of the sites of IL-1 synthesis and patterns of intracellular distribution provide further evidence for an extracellular release mechanism which is clearly distinct from the classical secretory pathway.

Blotting, Western↗
PubMed1992

Immunology of leishmaniasis.

Resolution of leishmanial infections requires the expansion of specific type 1 T helper cells that secrete or express on their membrane lymphokines capable of activating macrophages that contain these parasites to a microbicidal state. Specific CD8+ T cells, which are triggered during infection, also appear to play a role in protective immunity, possibly through their ability to secrete interferon-gamma. In the mouse model of infection with Leishmania major, the expansion of specific type 2 T helper cells exacerbates disease, an effect that appears to result from the properties of type 2 T helper derived lymphokines to deactivate macrophages and inhibit release of activating cytokines by type 1 T helper cells. In the mouse, destruction of intracellular Leishmania by activated macrophages depends upon the L-arginine-dependent production of nitrogen oxides. Molecules from the parasite that can induce, and are the target of, the protective T-cell response are being characterized.

Animals↗
PubMed1992

Lessons from Leishmania: a model for investigations of CD4+ subset differentiation.

Infection of inbred mice with Leishmania major remains the best model of human infection with visceralizing Leishmania that cause kala-azar. Immunologic investigations have correlated the outcome of disease with expansion of different subsets of CD4+ cells, designated Th1 and Th2. Although the capacity of fixed effector Th1 and Th2 populations to mediate the diverse outcomes of disease through the release of soluble cytokines, particularly IFN-gamma and IL-4, has been demonstrated, the mechanisms by which these subsets become established during infection have not been delineated. This review focuses on known features of CD4+ differentiation using other experimental models, and proposes that genetic susceptibility to Leishmania can occur if the host has a Th2 precursor cell in the memory configuration prior to the time of exposure to organisms, perhaps in response to cross-reactive self-peptides. The hypothesis can explain a number of puzzling observations in both murine and human disease due to these organisms and makes several predictions amenable to experimental testing.

Animals↗
PubMed1991

Reconstitution of Leishmania immunity in severe combined immunodeficient mice using Th1- and Th2-like cell lines.

Leishmania major disseminates in genetically susceptible BALB/c mice to cause fatal disease. Progressive infection has been linked to the failure of parasite-specific Th1, IFN-gamma-producing, CD4+ T lymphocytes to expand and direct macrophage activation and control of intracellular parasitism. In contrast, Th2 CD4+ cell expansion accompanies disease progression. Immunomodulation using CD4 cell depletion at the time of infection results in control of infection and Th1 CD4+ cell expansion. A Th1-like cell line, H1A, was established from the draining lymph nodes of an anti-CD4-pretreated BALB/c mouse infected with L. major, H1A was CD4, TCR(+)-alpha/beta, and released IL-2 and IFN-gamma in response to parasite Ag. A Th2-like cell line, U1A, was established from the lymph node cells of an infected BALB/c mouse that was also CD4, TCR(+)-alpha/beta but released IL-4 and IL-5 after stimulation. Mice with severe combined immunodeficiency were reconstituted with H1A and U1A before infection with L. major. Non-reconstituted mice were unable to restrict parasite growth. Mice reconstituted with H1A healed infection, whereas mice reconstituted with U1A suffered exacerbation of disease. Analysis of spleen cells by flow cytometry confirmed the reconstitution of CD4+ cells in both instances, and stimulation with mitogen established that the lymphokine profile of the donor cells had been maintained during 6 to 8 wk of infection. Histologic analysis of the lesions confirmed migration of donated cells to sites of infection. Neutralization of IFN-gamma in H1A-reconstituted mice and IL-4 in U1A-reconstituted mice reversed the disease phenotype mediated by the two cell lines. These data demonstrate the capacity of CD4+ T cells alone to modulate both positively and negatively the course of leishmaniasis in a lymphokine-dependent manner.

Animals↗
PubMed1991

Production of interferon gamma, interleukin 2, interleukin 4, and interleukin 10 by CD4+ lymphocytes in vivo during healing and progressive murine leishmaniasis.

The expression of interleukin (IL) 2, IL-4, IL-10, and interferon gamma (IFN-gamma) by lymphocyte subsets was examined during infection of resistant C57BL/6 and susceptible BALB/c mice with the protozoan parasite Leishmania major. CD4+ and CD8+ T lymphocytes and B lymphocytes were isolated from the lymph nodes draining infectious lesions, and their RNA was examined for lymphokine transcripts. Distinct patterns of CD4+ cell cytokine expression were apparent: C57BL/6 CD4+ cells contained IFN-gamma and IL-2 mRNA, whereas BALB/c CD4+ cells expressed IL-4 and IL-10 message. CD8+ cells contributed little lymphokine expression during disease, but B cells were a major source of IL-2 mRNA in both strains of mice. BALB/c mice made resistant by treatment with anti-CD4 antibody at the time of infection repopulated lymph nodes with CD4+ cells that expressed IL-2 and IFN-gamma. Protective treatment with anti-IL-4 antibody in vivo also resulted in the appearance of CD4+ cells with increased IFN-gamma and diminished IL-4 and IL-10 expression. These data establish CD4+ cells as the primary source of IFN-gamma in healing mice and of IL-4 and IL-10 during progressive infection and confirm that the spectral extremes of this disease are characterized by the presence of CD4+ cells expressing Th1 or Th2 phenotypes in vivo.

Animals↗