T-cell release of granulysin contributes to host defense in leprosy.

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major.

Previous studies with inhibitors of inducible nitric oxide synthase (iNOS) suggested that high-output production of nitric oxide (NO) is an important antimicrobial effector pathway in vitro and in vivo. Here, we investigated the tissue expression of iNOS in mice after infection with Leishmania major. Immunohistochemical staining with an iNOS-specific antiserum revealed that in the cutaneous lesion and draining lymph nodes (LN) of clinically resistant mice (C57BL/6), iNOS protein is found earlier during infection and in significantly higher amounts than in the nonhealing BALB/c strain. Similar differences were seen on the mRNA level as quantitated by competitive polymerase chain reaction. Anti-CD4 treatment of BALB/c mice not only induced resistance to disease, but also restored the expression of iNOS in the tissue. In situ, few or no parasites were found in those regions of the skin lesion and the draining LN which were highly positive for iNOS. By double labeling experiments, macrophages were identified as iNOS expressing cells in vivo. In the lesions of BALB/c mice, cells staining positively for transforming growth factor beta (TGF-beta), a potent inhibitor of iNOS in vitro, were strikingly more prominent than in C57BL/6, whereas no such difference was found for interleukin 4 or interferon gamma (IFN-gamma). In vitro, production of NO was approximately threefold higher in C57BL/6 than in BALB/c macrophages after stimulation with IFN-gamma. We conclude that the pronounced expression of iNOS in resistant mice is an important mechanism for the elimination of Leishmania in vivo. The relative lack of iNOS in susceptible mice might be a consequence of macrophage deactivation by TGF-beta and reduced responsiveness to IFN-gamma.

Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase.

Nitric oxide (NO) synthase (iNOS) is required for the resolution of acute cutaneous leishmaniasis in resistant C57BL/6 mice. As is the case in several other infections, the clinically cured host organism still harbors small amounts of live Leishmania major parasites. Here, we demonstrate lifelong expression of iNOS at the site of the original skin lesion and in the draining lymph node of long-term-infected C57BL/6 mice. iNOS activity in the lymph node was dependent on CD4+, but not on the CD8+ T cells. By double labeling techniques, iNOS and L. major were each found in macrophages (F4/80+, BM-8+, and/or MOMA-2+) and dendritic cells (NLDC-145+), but not in granulocytes or endothelial cells. In situ triple labeling of lymph node sections revealed that approximately 30-40% of the L. major foci were associated with iNOS-positive macrophages or dendritic cells. The majority of the L. major foci (60-70%), however, was located in areas that were negative for both iNOS and the macrophage and dendritic cell markers. In L. major-infected C57BL/6 mice, which had cured their cutaneous lesions, administration of L-N6-iminoethyl-lysine (L-NIL), a potent inhibitor of iNOS, led to a 10(4)-10(5)-fold increase of the parasite burden in the cutaneous and lymphoid tissue and caused clinical recrudescence of the disease. Persistent expression of iNOS and resumption of parasite replication after application of L-NIL was also observed in resistant C3H/HeN and CBA/J mice. We conclude that iNOS activity is crucial for the control of Leishmania persisting in immunocompetent hosts after resolution of the primary infection. Failure to maintain iNOS activity might be the mechanism underlying endogenous reactivation of latent infections with NO-sensitive microbes during phases of immunosuppression.

Molecular recognition of lipid antigens by T cell receptors.

The T cell antigen receptor (TCR) mediates recognition of peptide antigens bound in the groove of major histocompatibility complex (MHC) molecules. This dual recognition is mediated by the complementarity-determining residue (CDR) loops of the alpha and beta chains of a single TCR which contact exposed residues of the peptide antigen and amino acids along the MHC alpha helices. The recent description of T cells that recognize hydrophobic microbial lipid antigens has challenged immunologists to explain, in molecular terms, the nature of this interaction. Structural studies on the murine CD1d1 molecule revealed an electrostatically neutral putative antigen-binding groove beneath the CD1 alpha helices. Here, we demonstrate that alpha/beta TCRs, when transferred into TCR-deficient recipient cells, confer specificity for both the foreign lipid antigen and CD1 isoform. Sequence analysis of a panel of CD1-restricted, lipid-specific TCRs reveals the incorporation of template-independent N nucleotides that encode diverse sequences and frequent charged basic residues at the V(D)J junctions. These sequences permit a model for recognition in which the TCR CDR3 loops containing charged residues project between the CD1 alpha helices, contacting the lipid antigen hydrophilic head moieties as well as adjacent CD1 residues in a manner that explains antigen specificity and CD1 restriction.

Interferon-gamma release assay in the ascites: Early hint for diagnosis of abdominal tuberculosis.

We report on a 20-year-old woman with abdominal tuberculosis.Standard microbiological examination of ascites showed no acid-fast bacilli (AFB), and analysis for the Mycobacterium tuberculosis (MTB)-complex DNA by PCR was negative. However,the interferon-gamma release assay (IGRA) of the ascites was positive after specific stimulation with mycobacterial antigens(ESAT-6/CFP-10/TB7.7[p4]), indicating an infection with MTB.The diagnosis of tuberculosis was later confirmed by histology, MTB culture, and PCR analysis of MTB-complex DNA in tissue samples taken during laparoscopy. Thus, the IGRA of ascites may guide the decision to start active treatment for tuberculosis.

Differential effects of cytolytic T cell subsets on intracellular infection.

In analyzing mechanisms of protection against intracellular infections, a series of human CD1-restricted T cell lines of two distinct phenotypes were derived. Both CD4(-)CD8(-) (double-negative) T cells and CD8(+) T cells efficiently lysed macrophages infected with Mycobacterium tuberculosis. The cytotoxicity of CD4(-)CD8(-) T cells was mediated by Fas-FasL interaction and had no effect on the viability of the mycobacteria. The CD8(+) T cells lysed infected macrophages by a Fas-independent, granule-dependent mechanism that resulted in killing of bacteria. These data indicate that two phenotypically distinct subsets of human cytolytic T lymphocytes use different mechanisms to kill infected cells and contribute in different ways to host defense against intracellular infection.

An antimicrobial activity of cytolytic T cells mediated by granulysin.

Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

Induction of direct antimicrobial activity through mammalian toll-like receptors.

The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

Isolation of mycobacterium-reactive CD1-restricted T cells from patients with human immunodeficiency virus infection.

Because CD1-restricted T cells lack CD4 but produce IFN-gamma in response to nonpeptide mycobacterial antigens, they could play a unique role in immunity to tuberculosis. We studied CD1-restricted T cells in the context of HIV infection by expanding CD4(-) T cell lines from 10 HIV-infected patients. Upon stimulation with Mycobacterium tuberculosis antigen or upon exposure to macrophages infected with M. tuberculosis, these T cell lines proliferated, produced IFN-gamma, and showed cytolytic T cell (CTL) activity against macrophages pulsed with mycobacterial antigen, findings consistent with a protective role against M. tuberculosis. Anti-CD1b antibodies abrogated T cell proliferation, IFN-gamma production, and CTL activity, demonstrating that these T cells are CD1 restricted. IFN-gamma production in response to M. tuberculosis was enhanced by antitransforming growth factor-beta in 8/10 lines, and by IL-15 in 2/10 lines. IFN-gamma production was augmented in a nonantigen-specific manner by IL-12 in 4/8 lines. When live HIV was cocultured with CD1-restricted T cell lines, p24 antigen and proviral DNA were not detected, indicating that the T cells were not infectable with HIV. Vaccination strategies aimed at activation and expansion of M. tuberculosis-reactive CD1-restricted T cells in HIV-infected patients may constitute a novel means to provide protection against tuberculosis, while minimizing the risk of enhancing HIV replication through stimulation of CD4(+) cells.

Cytotoxic T cell responses to intracellular pathogens.

Host defense against intracellular pathogens is thought to require cytotoxic T cells. Recent studies have investigated the impact of host cell lysis and cytokine production by cytotoxic T lymphocytes on the fate of intracellular pathogens. The identification of two mechanisms of lysis induced by cytotoxic T lymphocytes--the granule exocytosis pathway and the Fas-FasL interaction--have provided new insight into the role of cytotoxic T lymphocyters in immunity to infection.

T cell mediated immunity to Mycobacterium tuberculosis.

Recent advances in the characterization of the protective immune response to mycobacteria have highlighted the central role of phenotypically and functionally distinct subsets of T cells. These T cell subsets not only contribute to host defense by the secretion of macrophage-activating cytokines, but also by lysing the host cell. Besides releasing intracellular pathogens, which can then be taken up and killed by newly recruited macrophages, it has now been demonstrated that lysis of infected targets by one subset of cytolytic T cells can directly kill Mycobacterium tuberculosis.

2,4-Diamino-6-hydroxypyrimidine, an inhibitor of tetrahydrobiopterin synthesis, downregulates the expression of iNOS protein and mRNA in primary murine macrophages.

2,4-diamino-6-hydroxy-pyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, blocks the synthesis of tetrahydrobiopterin (BH4), which is a known cofactor of inducible nitric oxide synthase (iNOS). Previously, DAHP was shown to suppress the production of nitric oxide by cytokine-activated fibroblasts, smooth muscle cells or endothelial cells which could be attributed to its function as a cofactor antagonist. Here, we demonstrate that in interferon-gamma-activated murine peritoneal macrophages DAHP suppresses the expression of iNOS mRNA and protein in a BH4-independent manner and, thus, acts by a novel mechanism.

L-N6-(1-iminoethyl)-lysine potently inhibits inducible nitric oxide synthase and is superior to NG-monomethyl-arginine in vitro and in vivo.

L-N6-(1-iminoethyl)-lysine is a novel inhibitor of nitric oxide (NO) synthase, which similar to aminoguanidine but unlike NG-monomethyl-L-arginine is 30-fold more selective for the inducible than for the constitutive isoform of the enzyme. Here, we characterized this inhibitor for the first time in intact cells and during infection of mice with a NO-sensitive parasite (Leishmania major). L-N6-(1-iminoethyl)-lysine potently inhibited the activity of inducible NO-synthase in primary macrophages. After stimulation by interferon-gamma the IC50 of L-N6-(1-iminoethyl)-lysine was 0.4 +/- 0.1 mu M and 10- or 30-fold lower than that of NG-monomethyl-L-arginine or aminoguanidine, respectively. In vivo, L-N6-(1-iminoethyl)-lysine (0.4-9 mM in the drinking water) suppressed inducible NO-synthase activity and caused a dramatic exacerbation of leishmaniasis, despite a counterregulatory increase of inducible NO-synthase protein in the tissue. In contrast, considerably higher concentrations of NG-monomethyl-L-arginine (20-50 mM) were required in order to achieve comparable effects. NG-monomethyl-L-arginine, but not L-N6-(1-imino-ethyl)-lysine led to weight loss, reduced water and food consumption. We conclude that L-N6-(1-iminoethyl)-lysine should be used instead of NG-monomethyl-L-arginine for potent suppression of inducible NO-synthase in vitro and in vivo.

Cytotoxic T lymphocytes in resistance to tuberculosis.

Recent experimental evidence has suggested T cells recognizing antigens in the context of both classical MHC class I and nonclassical class I-like molecules contribute to protective responses against Mycobacterium tuberculosis (MTB) infection. Our aims were to characterize both types of T cells, and to explore the basis of communication between the tubercle bacilli and the MHC class I pathway of the host macrophage. A model system was developed using exogenously added ovalbumin as a surrogate antigen to study presentation by MTB-infected macrophages. Viable, virulent MTB and closely related mycobacterial species facilitated the presentation of ovalbumin on MHC class I molecules to CD8+ cytolytic T cells that was dependent upon the cytosolic transport of peptides, implying communication between the MTB phagosome and the host cell cytoplasm. MHC class I presentation of soluble antigens was mimicked by Listeria monocytogenes, which grows within the host cell cytoplasm, as well as its purified hemolysin. We have also characterized T cells that recognize nonpeptide MTB antigens presented by CD1 molecules. CD1-restricted T cells demonstrated to lyse macrophages infected with virulent MTB were divided into distinct subsets based on surface phenotype (CD4-CD8- versus CD8-) and cytotoxicity mechanism (Fas receptor-mediated versus granule exocytosis). A functional consequence of these two mechanisms was observed that while both subsets lysed infected macrophages, only those T cells utilizing the granule exocytosis pathway were able to reduce viability of intracellular MTB.

Most-favored-nation clauses and monopsonistic power: an unhealthy mix?

Most-favored-nations clauses appear in health insurance contracts allegedly to prevent price discrimination by health care providers among competing insurers. In fact, use of these provisions often works to exclude competitors from the health insurance market. This Note examines the antitrust implications of most-favored-nations clauses as used in the health insurance industry.

Measuring the killing of intracellular pathogens: Leishmania.

Macrophages are professional phagocytes serving as a first line of defence against pathogenic organisms. Macrophages are equipped with efficient effector functions to kill invading microorganisms. The first important mechanism of macrophage host-defence is phagocytosis of pathogens. Subsequently, internalized pathogens are targeted for destruction in maturating phagolysosomal compartments. This process is mediated by lysosomal proteases and an acidified compartment. To investigate macrophages' killing potential in this chapter, we describe an assay based on human primary cells infected with the obligatory intracellular parasite Leishmania. For this pathogen the macrophage has a dual role. The parasite can use macrophages for its intracellular multiplication, but at the same time host macrophages, upon stimulation, can kill the parasite.

Immunological control of tuberculosis: role of tumour necrosis factor and more.

Therapy of autoimmune diseases with tumour necrosis factor (TNF) neutralising agents has provided a unique opportunity to learn about the significance of TNF in the maintenance of latent bacterial infections in humans. The remarkably high incidence of tuberculosis in patients treated with TNF antagonists raises the intriguing question about the physiological role of TNF in maintaining the lifelong latency of tubercle bacilli in granulomas in infected patients. Basic research during the past decade(s) combined with thoughtful observations in human subjects with tuberculosis and autoimmune diseases has provided several potential explanations for the recurrence of tuberculosis if TNF supply is withdrawn. TNF is involved in at least four key functions that contribute towards beneficial effects on the symptoms of autoimmune disorders on the one hand, and the attenuation of immune responses against Mycobacterium tuberculosis on the other hand. These are outlined in this review: induction of apoptosis, maturation of dendritic cells, activation of antimicrobial activity in macrophages, and orchestration of leucocyte movement.

Cytolytic T cells in the immune response to mycobacterium tuberculosis.

Cytolytic T cells (CTL) are of paramount importance in immune defense against tumors and viruses. Work over the past decade has revealed that lysis of infected cells is also involved in protective immunity to bacteria and parasites, including Mycobacterium tuberculosis. Experiments involving gene-deleted mice and the characterization of CTL lines derived from tuberculosis patients suggest an important role of CTL in immunity to tuberculosis. More recently, the identification of an effector pathway of human CTL provided evidence for direct antimicrobial activity of CTL. This pathway involves the combined action of the pore-forming perforin and the antibacterial granulysin, both expressed in the granules of CTL. Granulysin binds to the bacterial cell surface, thereby disrupting the membrane and causing osmotic lysis. The relevance of this pathway for protection against intracellular pathogens is suggested by the expression of high amounts of granulysin in tissue from patients with tuberculoid leprosy, which are able to contain the spread of the bacilli. These findings support the current concept of designing novel vaccination strategies which elicit not only CD4 + T helper cells, but also CD8 + CTL with direct antibacterial activity.

IL-10 converts human dendritic cells into macrophage-like cells with increased antibacterial activity against virulent Mycobacterium tuberculosis.

Dendritic cells (DC) are unique in their ability to initiate a primary immune response by the presentation of soluble Ags to T cells. Recent studies have shown that DC also phagocytose particulate Ags including the intracellular pathogen Mycobacterium tuberculosis. However, it is not known whether DC contain the growth of intracellular organisms or allow unlimited replication. To address this question, we infected human DC with a virulent strain of M. tuberculosis and monitored the intracellular growth. The bacteria grew two orders of magnitude within 7 days of culture. Among cytokines known to modulate mycobacterial growth particularly in murine macrophages (TNF-alpha, IFN-gamma, TGF-beta, IL-4), only IL-10 modulated the growth in human DC. This effect was specific for immature dendritic cells, as IL-10 did not induce growth inhibition in human macrophages. In searching for the mechanism of growth inhibition, we found that IL-10 induces the down-regulation of the DC marker CD1, while the macrophage marker CD14 was up-regulated. Functionally, IL-10-treated cells had a reduced capacity to induce an alloresponse, but phagocytic uptake of M. tuberculosis was more efficient. We also show that DC are inferior to macrophages in containing mycobacterial growth. These findings show that IL-10 converts DC into macrophage-like cells, thereby inducing the growth inhibition of an intracellular pathogen. At the site of a local immune response, such as a tuberculous granuloma, IL-10 might therefore participate in the composition of the cellular microenvironment by affecting the maturity and function of DC.