CD8+ T cell response to Mls-1a determinants involves major histocompatibility complex class II molecules.

Recent studies indicate that both CD4+ and CD8+ T lymphocytes proliferate in vitro in response to Mls-1a-encoded determinants. Using both immunogenetic and antibody blocking approaches we show here that Mls-1a responses of both subsets require expression of major histocompatibility complex (MHC) class II molecules (I-A and/or I-E) by the stimulator cells. Furthermore, CD8+ T cell responses to Mls-1a/class II MHC do not require (and are in fact inhibited by) the presence of functional CD8 molecules. Taken together, our data underscore the dramatic differences between CD8+ T cell responses to conventional peptide antigens as opposed to "superantigens" such as Mls-1a.

CD8+ T cells respond clonally to Mls-1a-encoded determinants.

T cell responses to the product of the minor lymphocyte stimulatory locus Mls-1a involve the selective use of TCR V beta domains (especially V beta 6 and V beta 8.1) and are generally considered to be restricted to the CD4+ mature subset. We show here that CD8+ (presumably MHC class I-restricted) T cells bearing V beta 6 or V beta 8.1 also respond preferentially to Mls-1a determinants either in vitro (in mixed leukocyte cultures) or in vivo (in an adoptive transfer system). In vitro responses of both CD4+ V beta 6+ and CD8+ V beta 6+ cells to Mls-1a were dependent upon the MHC haplotype of the stimulator cells, with I-E+ (H-2d or H-2k) alleles being much more stimulatory than I-E- (H-2q). These data strengthen the analogy between Mls gene products and other MHC class II-dependent superantigens such as the bacterial enterotoxins.

Distinct fates of self-specific T cells developing in irradiation bone marrow chimeras: clonal deletion, clonal anergy, or in vitro responsiveness to self-Mls-1a controlled by hemopoietic cells in the thymus.

Elimination of potentially self-reactive T lymphocytes during their maturation in the thymus has been shown to be a major mechanism in accomplishing self-tolerance. Previous reports demonstrated that clonal deletion of self-Mls-1a-specific V beta 6+ T lymphocyte is controlled by a radiosensitive I-E+ thymic component. Irradiation chimeras reconstituted with I-E- bone marrow showed substantial numbers of mature V beta 6+ T cells despite host Mls-1a expression. Analysis of the functional properties of such chimeric T cells revealed a surprising variability in their in vitro reactivity to host Mls-1a, depending on the H-2 haplotype of stem cells used for reconstitution. In chimeras reconstituted with B10.S (H-2s) stem cells, mature V beta 6+ lymphocytes were present but functionally anergic to host-type Mls-1a in vitro. In contrast, in chimeras reconstituted with B10.G (H-2q) bone marrow, nondeleted V beta 6+ cells were highly responsive to Mls-1a in vitro. These findings suggest that clonal anergy of V beta 6+ cells to self-Mls-1a may be controlled by the affinity/avidity of T cell receptor interactions with bone marrow-derived cells in the thymus depending on the major histocompatibility complex class II molecules involved. Furthermore, chimeras bearing host (Mls-1a)-reactive V beta 6+ cells did not differ clinically from those with anergic or deleted V beta 6+ cells and survived more than one year without signs of autoimmune disease. Interestingly, their spleen cells had no Mls-1a stimulatory capacity in vitro. Therefore, regulation at the level of antigen presentation may be an alternative mechanism for maintenance of tolerance to certain self-antigens such as Mls-1a.

Germinal center formation and local immunoglobulin E (IgE) production in the lung after an airway antigenic challenge.

Airway inflammation plays a central role in the pathogenesis of asthma. However, the precise contribution of all cell types in the development and maintenance of airway hyperreactivity and histopathology during allergic inflammation remains unclear. After sensitization of mice in the periphery, challenge by multiple intratracheal (i.t.) instillations of ovalbumin (OVA) results in eosinophilia, mononuclear cell infiltration, and airway epithelial changes analogous to that seen in asthma (Blyth, D.I., M.S. Pedrick, T.J. Savage, E.M. Hessel, and D. Fattah. 1996. Am. J. Respir. Cell Mol. Biol. 14:425-438). To investigate further the nature of the cellular infiltrate, lungs from OVA-versus saline-treated mice were processed for histology and immunohistochemistry. One of the most striking features observed was the formation of germinal centers within the parenchyma of the inflamed lungs. In addition, follicular dendritic cells (FDCs) bearing OVA on their plasma membranes appeared and, adjacent to these sites, OVA-specific IgG1-, IgE-, and IgA-producing plasma cells emerged. To confirm that antigen-specific immunoglobulins (Ig) were being produced within the parenchyma, plasma cell number and antibody production were quantitated in vitro after isolation of cells from the lung. These assays confirmed that the isotypes observed in situ were a secreted product. As IgE-dependent mechanisms have been implicated as being central to the pathogenesis of bronchial asthma, airway hyperresponsiveness was evaluated. The mice undergoing lung inflammation were hyperresponsive, while the control group remained at baseline. These data demonstrate that antigen-driven differentiation of B cells via induction of an FDC network and germinal centers occurs in the parenchyma of inflamed lungs. These germinal centers would then provide a local source of IgE-secreting plasma cells that contribute to the release of factors mediating inflammatory processes in the lung.

A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock.

CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4(-/-)) mice by gene targeting. CCR4(-/-) mice developed normally. Splenocytes and thymocytes isolated from the CCR4(-/-) mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1alpha. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4(-/-) mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4(+/+) mice. After high dose LPS treatment, serum levels of tumor necrosis factor alpha, interleukin 1beta, and MIP-1alpha were reduced in CCR4(-/-) mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4(-/)- mice by flow cytometry also revealed a significant decrease in the F4/80(+) cell population. This may reflect a defect in the ability of the CCR4(-/-) macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.

Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis.

Two distinct IL-18 neutralizing strategies, i.e. a rabbit polyclonal anti-mouse IL-18 IgG and a recombinant human IL-18 binding protein (rhIL-18BP), were used to treat collagen-induced-arthritic DBA/1 mice after clinical onset of disease. The therapeutic efficacy of neutralizing endogenous IL-18 was assessed using different pathological parameters of disease progression. The clinical severity in mice undergoing collagen-induced arthritis was significantly reduced after treatment with both IL-18 neutralizing agents compared to placebo treated mice. Attenuation of the disease was associated with reduced cartilage erosion evident on histology. The decreased cartilage degradation was further documented by a significant reduction in the levels of circulating cartilage oligomeric matrix protein (an indicator of cartilage turnover). Both strategies efficiently slowed disease progression, but only anti-IL-18 IgG treatment significantly decreased an established synovitis. Serum levels of IL-6 were significantly reduced with both neutralizing strategies. In vitro, neutralizing IL-18 resulted in a significant inhibition of TNF-alpha, IL-6, and IFN-gamma secretion by macrophages. These results demonstrate that neutralizing endogenous IL-18 is therapeutically efficacious in the murine model of collagen-induced arthritis. IL-18 neutralizing antibody or rhIL-18BP could therefore represent new disease-modifying anti-rheumatic drugs that warrant testing in clinical trials in patients with rheumatoid arthritis.

Interleukin-18/interleukin-18 binding protein signaling modulates atherosclerotic lesion development and stability.

Interleukin (IL)-18 is the interferon-gamma-inducing factor and has other proinflammatory properties. The precise role of IL-18 in immunoinflammatory diseases remains poorly understood. In this study, we show that in vivo electrotransfer of an expression-plasmid DNA encoding for murine IL-18 binding protein (BP) (the endogenous inhibitor of IL-18) prevents fatty streak development in the thoracic aorta of apoE knockout mice and slows progression of advanced atherosclerotic plaques in the aortic sinus. More importantly, transfection with the IL-18BP plasmid induces profound changes in plaque composition (decrease in macrophage, T cell, cell death, and lipid content and increase in smooth muscle cell and collagen content) leading to a stable plaque phenotype. These results identify for the first time a critical role for IL-18/IL-18BP regulation in atherosclerosis and suggest a potential role for IL-18 inhibitors in reduction of plaque development/progression and promotion of plaque stability. The full text of this article is available at http://www.circresaha.org.

Expression of interleukin-18 in human atherosclerotic plaques and relation to plaque instability.

BACKGROUND: Interleukin (IL)-18 is a potent proinflammatory cytokine with potential atherogenic properties. Its expression and role in atherosclerosis, however, are unknown. METHODS AND RESULTS: In the present study, we examined stable and unstable human carotid atherosclerotic plaques retrieved by endarterectomy for the presence of IL-18 using reverse transcription-polymerase chain reaction (PCR), Western blot, and immunohistochemical techniques. IL-18 was highly expressed in the atherosclerotic plaques compared with control normal arteries and was localized mainly in plaque macrophages. IL-18 receptor was also upregulated in plaque macrophages and endothelial cells, suggesting potential biological effects. To examine the role of IL-18 in atherosclerosis, we determined the relation between IL-18 mRNA expression and signs of plaque instability using real-time quantitative PCR. Interestingly, significantly higher levels of IL-18 mRNA were found in symptomatic (unstable) plaques than asymptomatic (stable) plaques (P<0.01). CONCLUSIONS: These results suggest, for the first time, a major role for IL-18 in atherosclerotic plaque destabilization leading to acute ischemic syndromes.

Immunoprecipitation of insulin receptors by antibodies against Class 1 antigens of the murine H-2 major histocompatibility complex.

Insulin receptors from C57BL/6J mouse (H-2b) liver membranes were specifically labeled with 125I-photo-reactive insulin by UV irradiation. Membranes were solubilized and the capacity of various antibodies reacting with the major histocompatibility complex to immunoprecipitate insulin receptors was tested. About 5% of the labeled receptors were immunoprecipitated by a conventional mouse antiserum against H-2b histocompatibility antigens and by a monoclonal antibody against Class 1 antigens of the H-2b haplotype (Kb and Db). No immunoprecipitation was obtained with a monoclonal antibody against Class 2 antigens of I-Ab or against Class 1 antigens of the H-2k haplotype. Insulin receptors can thus be specifically immunoprecipitated by antibodies against class I histocompatibility antigens.

The subunit structure of the insulin receptor and molecular interactions with major histocompatibility complex antigens.

Insulin receptors were labeled with 125I-photoreactive insulin (specifically labeling alpha-subunits) and by insulin-stimulated autophosphorylation (specifically labeling beta-subunits). The results show that the insulin receptor exists under different free and disulfide-linked combinations of alpha and beta subunits. Moreover, the insulin receptor is closely associated to class I antigens of the major histocompatibility complex to form a high molecular weight multi-molecular membrane complex.

Subunit arrangement of insulin receptors in hepatoma cells.

Insulin receptors from rat hepatoma cells were studied by the three following methods. Firstly, the alpha subunit (Mr 130000) was labelled using a 125I-photoreactive insulin analogue and UV irradiation. Secondly, using phosphorylation of partially purified and immunoprecipitated receptors with [gamma-32P]ATP, the beta subunit (Mr 95000) was labelled. Thirdly, both alpha and beta subunits were labelled by surface iodination catalysed by lactoperoxidase followed by cell solubilization and immunoprecipitation of the receptor with anti-receptor antibodies. The results show that the native insulin receptor exists under different forms: free alpha and beta subunits and the following combinations of disulphide-linked oligomers: alpha beta, alpha 2, alpha 2 beta and alpha 2 beta 2. In addition, it appears that there is at least one insulin binding site per alpha subunit, and that the alpha and beta subunits may be in close physical association in the plasma membrane even when they are not linked by disulphide bonds. In intact cells, only the alpha subunit is sensitive to extracellular proteases that cleave preferentially the region of the alpha subunit bearing the sulphydryl groups responsible for the interchain binding.

Two yeast mutants defective in endocytosis are defective in pheromone response.

We have purified biosynthetically labeled alpha-factor secreted from transformed yeast alpha cells. This alpha-factor binds specifically to a cells and is internalized by a time-, temperature-, and energy-dependent process. alpha-factor is internalized in an intact form and then rapidly degraded. Two yeast mutants defective in the accumulation of an endocytotic marker, lucifer yellow CH, in the vacuole have been isolated. end1 accumulates invaginations of the plasma membrane, and end2, an internal membrane-bound organelle. One of these mutants, end1, is defective for internalization of alpha-factor. Both of these mutants are defective in pheromone response.

The physiology of murine germinal center reactions.

Germinal center responses are the mechanism that the immune system uses normally to generate high affinity antigen-specific B-cell receptors and secreted immunoglobulins. Genetically altered mice have provided powerful tools for dissecting the physiology of these germinal center responses. In this review, we have attempted to summarize information from various sources and interpret the new observations based on what was previously known. A section is included to review the basic anatomy of the relevant structures in lymph node and spleen. A summary of the mutant mice producing a phenotype where germinal center responses are altered is also furnished. This review is aimed at providing useful information to those working in this field as well as those wishing to understand more about in vivo immunology.

Internalization and recycling of insulin receptors in hepatoma cells. Absence of regulation by receptor occupancy.

Insulin receptors of Fao hepatoma cells were labelled with a 125I-labelled photoreactive insulin analogue or by surface iodination catalysed by lactoperoxidase. Cells were then incubated at 37 degrees C, and the cellular localization of the labelled receptors was assessed by limited exposure of intact cells to trypsin. The results show that: (1) photolabelled insulin-receptor complexes are internalized and recycled in Fao hepatoma cells; (2) the dynamics of photolabelled insulin receptors (internalization and recycling) is similar before and after down-regulation; (3) the unoccupied receptors labelled by surface iodination are internalized and recycled similarly to covalent insulin-receptor complexes; (4) insulin does not induce internalization of surface-iodinated insulin receptors. We conclude that internalization and recycling of insulin receptors are independent of receptor occupancy by insulin in Fao hepatoma cells.

The viral superantigen Mls-1a induces interferon-gamma secretion by specifically primed CD8+ cells but fails to trigger cytotoxicity.

Superantigens can be operationally defined by their ability to stimulate CD4+ and CD8+ T cells via the T cell receptor beta chain variable domain (TcR V beta). We show here that effector functions of CD8+ T cells specific for superantigens differ depending upon the nature of the superantigen involved. Hence, activated CD8+ T cells bearing TcR V beta specific for the superantigen Mls-1a [encoded in the open reading frame of the 3' long terminal repeat of endogenous mouse mammary tumor virus (MMTV)] are unable to lyse Mls-1a-bearing target cells despite the fact that they release interferon-gamma (IFN-gamma) upon Mls-1a stimulation. In contrast CD8+ T cells specific for the exogenous superantigen staphylococcal enterotoxin B (SEB) readily mediate both lysis and IFN-gamma secretion when exposed to SEB-bearing target cells. This dissociation between lysis and IFN-gamma production by Mls-1a-specific CD8+ T cells is independent of the initial stimulus used for activation and appears not to be simply explained by a low Mls-1a determinant density. We suggest that this phenomenon reflects differing TcR affinity thresholds for lymphokine secretion and cytolysis. Such differences may be exploited by retroviruses such as MMTV in order to escape immunosurveillance.

The involvement of an ATP-gated ion channel, P(2X1), in thymocyte apoptosis.

In the immune system, apoptosis is involved in intrathymic elimination of self-reactive thymocytes and in peripheral T cell tolerance to exogenous antigens. Here, we describe the role in T cell apoptosis of P(2x1), a nonselective cation channel activated by ATP. P(2X1) molecules are up-regulated in thymocytes during dexamethasone-induced apoptosis, and antagonists to these receptors protect thymocytes from cell death. Moreover, P(2X1) mRNA and protein levels increase in thymocytes induced to die in vivo by the superantigen staphylococcal enterotoxin B. In contrast, T cells undergoing apoptosis in the periphery do not express P(2X1). The demonstration that P(2X1) ion channels play a role in the apoptosis of thymocytes but not peripheral T cells illustrates a novel mechanism contributing to thymocyte cell death and opens new possibilities for investigating clonal deletion in the thymus.

Modulation of T-cell response to phospholipase A2 and phospholipase A2-derived peptides by conventional bee venom immunotherapy.

BACKGROUND: Immunologic mechanisms of desensitization are still incompletely understood. Safer methods of immunotherapy with reduced risks of anaphylaxis need to be developed. OBJECTIVE: To study the effects of conventional venom immunotherapy (VIT) on phospholipase A2(PLA2)-specific T cells and on T-cell reactivity to short and long synthetic peptides that map the PLA2 molecule. METHOD: Proliferation of a CD4+ cell-enriched peripheral blood mononuclear cell fraction and cytokine secretion by T cell lines from patients hypersensitive to bee venom and undergoing VIT in response to PLA2 and PLA2 synthetic peptides were measured. RESULTS: T-cell proliferation in response to three synthetic peptides, 40 to 60 amino acids long and mapping the entire PLA2 molecule with an overlap of 10 residues (1 to 59, 51 to 99, and 90 to 134) steadily increased during the first 14 weeks of VIT corresponding to the treatment period with incremental doses of antigen. These results are in contrast to the low proliferation indices obtained with short (15 amino acid-long) peptides, and the inability to characterize the immunodominant region of the molecule with short peptides. At the end of VIT (after 3 to 5 years), there was correspondingly, a marked decrease in T cell responsiveness to PLA2 and to its long synthetic peptides. This response was paralleled by a shift in the pattern of cytokine secretion by T cell lines from a T(H0)-type to a T(H1)-type pattern. CONCLUSION: After a transient increase in T-cell proliferation, late VIT was characterized by T-cell hyporesponsiveness to allergen and by modulation of cytokine secretion from a T(H0)-type to a T(H1)-type pattern. Because of their capacity to recruit multiple T-cell epitopes, long peptides mapping the entire PLA2 molecule appear to be efficient T cell stimulators and may represent potential candidates for peptide immunotherapy.

Lethal Mycobacterium bovis Bacillus Calmette Guérin infection in nitric oxide synthase 2-deficient mice: cell-mediated immunity requires nitric oxide synthase 2.

The role of nitric oxide (NO) in Mycobacterium bovis Bacillus Calmette Guerin (BCG) infection was investigated using nitric oxide synthase 2 (nos2)-deficient mice, because NO plays a pivotal protective role in M. tuberculosis infection. We demonstrate that nos2-deficient mice were unable to eliminate BCG and succumbed within 8 to 12 weeks to BCG infection (10(6) CFU) with cachexia and pneumonia, whereas all infected wild-type mice survived. The greatest mycobacterial loads were observed in lung and spleen. Nos2-deficient mice developed large granulomas consisting of macrophages and activated T cells and caseous necrotic lesions in spleen. The macrophages in granulomas from nos2-deficient mice had reduced acid phosphatase activities, suggesting that NO is required for macrophage activation. The absence of NOS2 affected the cytokine production of the Th1 type of immune response, except IL-18. Serum amounts of IL-12p40 were increased and IFN-gamma was decreased compared with wild-type mice. The lack of NOS2 resulted in an overproduction of TNF, observed throughout the infection period. Additionally, TNFR1 and TNFR2 shedding was altered compared with wild-type mice. Up-regulation of TNF may be compensatory for the lack of NOS2. The late neutralization of TNF by soluble TNF receptors resulted in heightened disease severity and accelerated death in nos2-deficient mice but had no effect in wild-type mice. In conclusion, the inability of nos2-deficient mice to kill M. bovis BCG resulted in an accumulation of mycobacteria with a dramatic activation of the immune system and overproduction of pro-inflammatory cytokines, which resulted in death.

Delineation of PLA2 epitopes using short or long overlapping synthetic peptides: interest for specific immunotherapy.

BACKGROUND: Venom immunotherapy is definitely indicated in severe systemic anaphylactic reactions to bee stings, but is not devoided of risks of anaphylaxis. Safer methods of immunotherapy need to be developed. OBJECTIVE: To delineate phospholipase A2 T-cell epitopes using short 15mer vs long 40-60mer overlapping peptides, and to approach the potential interest of a venom immunotherapy based on the use of long peptides (1-60, 51-99, 90-134) mapping the whole phospholipase A2 molecule vs a restricted number of immunodominant epitopes. METHODS: Proliferation of a CD8+ T cell depleted peripheral blood mononuclear cell fraction and short-term T-cell lines from unselected bee venom hypersensitive patients in response to phospholipase A2 synthetic peptides. RESULTS: Whereas T-cell proliferation to 15mer overlapping peptides was weak, T-cell response to long overlapping peptides was in contrast vigorous in all patients, mostly directed to C-terminal peptide 90-134. Our results did not support the concept of rare dominant T-cell epitopes, and disclosed T-cell responses to multiple epitopes in several patients. No significant IgE-binding to long overlapping peptides was detected except in one patient against peptide 90-134. CONCLUSION: 15mer peptides might not be sensitive enough to fully delineate all potential T-cell epitopes scattered along the allergen. Since they do not bind IgE in vitro or only weakly, and taking into account a T-cell response frequently directed to multiple epitopes, long overlapping peptides may represent ideal tools for immunotherapy.