Cytokine fields and the polarization of the immune response.

Certain cells that participate in the immune response are known to become polarized in their production of cytokines. It is postulated that, after initial polarization at the site of antigenic encounter, the different types of cell arriving at this site are induced to conform to the local cytokine field, implying that they share common regulatory circuits. As they migrate, these cells might, in turn, spread the particular cytokine field. Therefore, the field is 'infectious' in nature. Propagation of the cytokine field must be regulated somehow. The invasion of the cytokine field into an organ or the entire body could have major immunological consequences.

Fruit-specific lectins from banana and plantain.

One of the predominant proteins in the pulp of ripe bananas (Musa acuminata L.) and plantains (Musa spp.) has been identified as a lectin. The banana and plantain agglutinins (called BanLec and PlanLec, respectively) were purified in reasonable quantities using a novel isolation procedure, which prevented adsorption of the lectins onto insoluble endogenous polysaccharides. Both BanLec and PlanLec are dimeric proteins composed of two identical subunits of 15 kDa. They readily agglutinate rabbit erythrocytes and exhibit specificity towards mannose. Molecular cloning revealed that BanLec has sequence similarity to previously described lectins of the family of jacalin-related lectins, and according to molecular modelling studies has the same overall fold and three-dimensional structure. The identification of BanLec and PlanLec demonstrates the occurrence of jacalin-related lectins in monocot species, suggesting that these lectins are more widespread among higher plants than is actually believed. The banana and plantain lectins are also the first documented examples of jacalin-related lectins, which are abundantly present in the pulp of mature fruits but are apparently absent from other tissues. However, after treatment of intact plants with methyl jasmonate, BanLec is also clearly induced in leaves. The banana lectin is a powerful murine T-cell mitogen. The relevance of the mitogenicity of the banana lectin is discussed in terms of both the physiological role of the lectin and the impact on food safety.

Increased protein synthesis after T cell activation in presence of cyclosporin A.

BACKGROUND: The immunosuppressive drug, cyclosporin A (CsA), blocks immune responses by inhibiting the calcineurin-dependent dephosphorylation of the nuclear factor of activated T cells (NFAT). We have previously reported that T cells activated in presence of CsA exhibit particular properties. In our study, we have tested the hypothesis that T cells activated in presence of CsA display a differential pattern of gene expression. METHODS: T lymphocytes were activated in vitro by Concanavalin A with or without CsA. The cells were: (1) pulsed with 35S-methionine to label the newly synthesized proteins that in turn were revealed by 2D-gel electrophoresis; (2) analyzed by flow cytometry for activation markers expression; and (3) examined by gel electrophoresis for early tyrosine phosphorylation events. RESULTS: The proteomic patterns of T lymphocytes activated by Concanavalin A, with or without CsA, were compared. In keeping with the well-known effect of the immunosuppressor, many polypeptides were not found in its presence. Remarkably, several newly synthesized polypeptides were detected only when activation was carried out in presence of CsA. In addition, immunologically relevant proteins, such as CD44 and CD69, escape CsA-inhibitory action. Furthermore, CsA did not modify the early protein tyrosine phosphorylation events resulting from T cell triggering. CONCLUSIONS: The present data show that the effect of CsA on protein synthesis is more complex than anticipated. Signaling provided by T cell activation and the blockade of the calcineurin-dependent pathway by CsA results in an altered program of gene expression.

Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants.

A novel plant lectin was isolated from salt-stressed rice (Oryza sativa L.) plants and partially characterized. The lectin occurs as a natural mixture of two closely related isoforms consisting of two identical non-covalently linked subunits of 15 kDa. Both isoforms are best inhibited by mannose and exhibit potent mitogenic activity towards T-lymphocytes. Biochemical analyses and sequence comparisons further revealed that the rice lectins belong to the subgroup of mannose-binding jacalin-related lectins. In addition, it could be demonstrated that the lectins described here correspond to the protein products of previously described salt-stress-induced genes. Our results not only identify the rice lectin as a stress protein but also highlight the possible importance of protein-carbohydrate interactions in stress responses in plants.

Crystal structure of Urtica dioica agglutinin, a superantigen presented by MHC molecules of class I and class II.

BACKGROUND: Urtica dioica agglutinin (UDA), a monomeric lectin extracted from stinging nettle rhizomes, is specific for saccharides containing N-acetylglucosamine (GlcNAc). The lectin behaves as a superantigen for murine T cells, inducing the exclusive proliferation of Vbeta8.3(+) lymphocytes. UDA is unique among known T cell superantigens because it can be presented by major histocompatibility complex (MHC) molecules of both class I and II. RESULTS: The crystal structure of UDA has been determined in the ligand-free state, and in complex with tri-acetylchitotriose and tetra-acetylchitotetraose at 1.66 A, 1.90 A and 1.40 A resolution, respectively. UDA comprises two hevein-like domains, each with a saccharide-binding site. A serine and three aromatic residues at each site form the principal contacts with the ligand. The N-terminal domain binding site can centre on any residue of a chito-oligosaccharide, whereas that of the C-terminal domain is specific for residues at the nonreducing terminus of the ligand. We have shown previously that oligomers of GlcNAc inhibit the superantigenic activity of UDA and that the lectin binds to glycans on the MHC molecule. We show that UDA also binds to glycans on the T cell receptor (TCR). CONCLUSIONS: The presence of two saccharide-binding sites observed in the structure of UDA suggests that its superantigenic properties arise from the simultaneous fixation of glycans on the TCR and MHC molecules of the T cell and antigen-presenting cell, respectively. The well defined spacing between the two binding sites of UDA is probably a key factor in determining the specificity for Vbeta8.3(+) lymphocytes.

Proteomic analysis of T cell activation in the presence of cyclosporin A: immunosuppressor and activator removal induces de novo protein synthesis.

Cyclosporin A (CsA), a fungal metabolite used in organ transplantation, blocks the immune responses by interfering with early activation signals preventing the induction of the IL2 gene. We have previously reported that the removal of the immunosuppressor provokes the transcription of the IL2 encoding gene. We have now investigated whether the transcription and translation of other genes accompanies this process. Withdrawal of CsA and Concanavalin A (ConA) from cultures of murine T cells activated by ConA in the presence of CsA leads to substantial changes in the pattern of radio-labelled proteins. A large number of polypeptides were synthesised de novo. In addition, a set of polypeptides detected prior to immunosuppressor elimination was not anymore synthesised. Finally, besides these qualitative changes, quantitative differences in terms of increased or decreased polypeptide abundance were also observed. The results demonstrate that activation in the presence of CsA has programmed the T cells to transcribe and translate a large number of genes, without further reactivation, once the immunosuppressor and the activator were removed.

The impact of immunosuppressive drugs on the analysis of T cell activation.

The discovery of the immunosuppressive properties of cyclosporin A (CSA) and its successful utilisation in organ transplantation was a milestone in clinics. CSA has revolutionised transplantation both in term of efficiency and quality-of-life of the patient. In addition, the analysis of the mode of action of CSA has been rewarding in the understanding the mechanisms leading to T lymphocytes activation. CSA binds to a family of cytosolic receptors, the cyclophilins, a highly conserved family of proteins. Once this complex is formed, a third protein, the calcineurin, is recruited. The calcineurin, a calcium-dependent phosphatase, loose its activity when complexed. Dephosphorylation of NFAT, a substrate of calcineurin is a mandatory step for its translocation to the nucleus where NFAT acts as a transactivator involved in the regulation of the genes encoding many cytokines. CSA preventing NFAT dephosphorylation blocks cytokine production this in turn allowing for a better engrafting. The resolution of the tertiary structure of CSA alone or complexed with cyclophilin and calcineurin has important implication in the modelling of new drugs devoid of its side effects. Indeed, the high incidence of cancer is one of the main problems linked to CSA utilisation. Recent data suggest that CSA may promote cancer inducing the transcription of the gene encoding transforming growth factor beta. Other molecules sharing with CSA its immunosuppressive activity were later described. Some of them, as FK506, have the some mode of action; others, as rapamycin, mycophenolate mofetil or leflunomide, act at different steps of T cell activation.

Major histocompatibility class I molecules present Urtica dioica agglutinin, a superantigen of vegetal origin, to T lymphocytes.

The Urtica dioica agglutinin (UDA) shares with the superantigens the property of activating T cell subsets bearing particular Vbeta segments of the TCR. However, UDA is a lectin capable of binding to many glycoproteins on cell membranes. The implication of MHC versus other glycoproteins in UDA presentation was presently studied. Using mutant mice lacking MHC class I (MHC-I), MHC class II (MHC-II) or both MHC antigens, we provided evidence that MHC-I and MHC-II molecules serve as UDA receptors. Presentation by either one of these molecules ensured similar T cell responses and co-stimulatory signals were mandatory for optimal T cell activation and proliferation both in MHC-I and MHC-II contexts. Remarkably, in the absence of MHC molecules, UDA could not be efficiently presented to T cells by other glycosylated proteins. Surface plasmon resonance studies were used to confirm the binding of UDA to MHC-I molecules using a fusion protein consisting of MHC-I domains and beta2-microglobulin. The results indicated that the interaction between UDA and MHC-I molecules implicated lectin-binding site(s) of UDA. Taken together, our data demonstrate that, in addition to MHC-II antigens, MHC-I molecules serve as an alternative ligand for UDA.

Administration to mouse of endotoxin from gram-negative bacteria leads to activation and apoptosis of T lymphocytes.

Lipopolysaccharide (LPS) from gramnegative bacteria is a well-known T cell-independent B lymphocyte mitogen and macrophage/monocyte activator. While the conventional view holds that LPS is ignored by T cells, we report here that administration of LPS to mice activates all B cells, but also engages most CD4 and CD8 T cells, as measured by the expression of the activation markers CD69 and CD25 and by size increase. T cells recruited in endotoxin-treated mice showed, following in vitro stimulation by concanavalin A, altered patterns of cytokine production. In vivo, massive T cell apoptosis was evidenced in the days following LPS exposure. The present observation may contribute novel insights into the mechanisms of endotoxin shock and of the immunological consequences of gram-negative infections.

Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance.

Previous studies have shown that the capacity to clear an attenuated strain of Salmonella typhimurium after the second week of infection varies widely among mouse strains. Bacterial clearance is mediated by CD4+ T cells and is regulated in part by the H-2 complex. The aim of the present study was to compare the patterns of cytokine mRNA expression in the spleens of C57BL/6 (H-2b) and CBA (H-2k) mice, which exhibit a low and a high rate of bacterial clearance, respectively. A transient increase in interleukin-12 (IL-12) mRNA levels was found in both mouse strains. Gamma interferon (IFN-gamma) gene expression was higher and more sustained in C57BL/6 than in CBA mice. No increase in IL-4 mRNA was detected. A transient increase in IL-10 mRNA was found in C57BL/6 mice. Separation of spleen cells into CD4+ and CD4- fractions showed that CD4+ T cells produced the bulk of IFN-gamma in both mouse strains and of IL-10 in C57BL/6 mice. Infection of H-2 congenic mice induced a higher level of IFN-gamma mRNA expression by CD4+ T cells in mice with a low rate of clearance (H-2b) than in mice with a high rate of clearance (H-2q). Treatment of infected C57BL/6 mice with anti-IFN-gamma or anti-CD4 monoclonal antibodies indicated that IFN-gamma participates in resistance in the early phase of infection, but not in bacterial clearance, and that CD4+ T cells mediate bacterial clearance during the 3rd week of infection. Taken together, these results suggest that defective bacterial clearance in H-2b mice is not linked to defective IFN-gamma production and that CD4+ T cells mediate bacterial clearance by an IFN-gamma-independent mechanism.

Isolation of a novel plant lectin with an unusual specificity from Calystegia sepium.

A novel plant lectin has been isolated from the rhizomes of Calystegia sepium (hedge bindweed) and partially characterized. The lectin is a dimeric protein composed of two identical non-covalently linked subunits of 16 kDa. Hapten inhibition studies indicate that the novel lectin is best inhibited by maltose and mannose and hence exhibits a sugar binding specificity that differs in some respects from that of all previously isolated plant lectins. Mitogenicity tests have shown that the Calystegia lectin is a powerful T-cell mitogen. Affinity purification of human, plant and fungal glycoproteins on immobilized C. sepium lectin demonstrates that this novel lectin can be used for the isolation of glycoconjugates from various sources. Moreover, it can be expected that by virtue of its distinct specificity, the new lectin will become an important tool in glycobiology.

Colchicum autumnale agglutinin activates all murine T-lymphocytes but does not induce the proliferation of all activated cells.

Plant lectins with mitogenic properties for T-lymphocytes have been particularly useful for the study of T-cell activation and effector functions. In the search for mitogenic lectins possessing activation features different from the ones associated with the already known mitogens, we found that an agglutinin isolated from Colchicum autumnale tubers, Colchicum autumnale agglutinin (CAA), possesses interesting properties. First, contrasting with the classical mitogens, CAA induces the proliferation of a fraction of the CD4+ and CD8+ mouse T-lymphocytes. Second, the CAA-induced proliferation requires MHC class II and CD4 molecules. Third, although only a fraction of T-cells enters into the cell cycle, all T-lymphocytes are activated and express high levels of the activation markers CD69 and CD44. Finally, CAA-stimulation is characterized by a particular pattern of the cytokine gene expression, reflected by the transcription of the IL2, IL5, and IFN-gamma genes, while the IL4 and IL10 genes remained silent. Taken together these data demonstrate that CAA activation does not conform to the pathway of T-cell triggering observed with classical mitogenes and represents a new tool for the analysis of T-cell activation.

Urtica dioica agglutinin, a V beta 8.3-specific superantigen, prevents the development of the systemic lupus erythematosus-like pathology of MRL lpr/lpr mice.

The V beta 8.3-specific superantigenic lectin Urtica dioica agglutinin (UDA) was used to delete the V beta 8.3+ T cells in MRL lpr/lpr mice. In contrast to the systemic lupus erythematosus-like pathology which progresses with age in the phosphate-buffered saline-injected MRL lpr/lpr controls, UDA-treated animals did not develop overt clinical signs of lupus and nephritis. The pathogenic T cell clones thus reside within the V beta 8.3+ T cell population, which includes an expanded T cell clone described previously. Finally, UDA alters the production of autoantibodies in a sex-dependent manner.

T cell activation by concanavalin A in the presence of cyclosporin A: immunosuppressor withdrawal induces NFATp translocation and interleukin-2 gene transcription.

Cyclosporin A (CSA), an immunosuppressive agent used in organ transplantation and to treat some autoimmune diseases, blocks the Ca2+-dependent steps involved in T cell receptor triggering leading to interleukin (IL)-2 production. Considering that the early steps of T cell activation are insensitive to CSA, we asked whether the initial activation achieved in presence of this immunosuppressor could affect the capacity of the T cell to respond to a mitogenic restimulation. We found that T cells activated by concanavalin A (ConA) for 48 h in the presence of CSA retain the capacity to proliferate in response to ConA once the immunosuppressor is removed. These cells are able to transcribe anew the IL-2 gene, without the requirement of new protein synthesis, and to up-regulate the alpha chain of the IL-2 receptor. Furthermore, we present the first direct evidence that the nuclear factor AP-1 is present in the nucleus of the T cells primed for 48 h in presence of CSA and that withdrawal of the immunosuppressor leads to the translocation of NFATp from the cytoplasm to the nucleus.

Opposite effects of interleukin-4 on memory T helper cell development depend on interleukin-2.

We previously reported that cyclosporin A (CSA) promotes the generation of T helper memory cells during antigenic priming of murine spleen cells in vitro. More recently, we have demonstrated that interleukin-2 (IL2) has a downmodulating effect on T helper memory cell generation. The present data address the role of the other T cell growth factor, IL4, upon induction of these cells. The data presented here show that IL4 can interfere with this process: addition of rIL4 to immunosuppressed priming cultures leads to a considerable decrease in the helper activity of the recovered cells. However, in standard cultures, in which IL2 is normally produced, no effect of IL4 on T helper memory cell generation was found. Addition of IL4 has important consequences for cytokines produced upon antigenic restimulation. In standard cultures, IL4 primes for cells expressing high levels of IL2 and IL4 mRNA. Strikingly, in immunosuppressed priming cultures, IL4 counterbalances the CSA-induced blockade of the IFN gamma gene. Taken together, our results suggest that the unique role of IL4 is to drive T helper memory precursors into an IL4 production differentiation pathway. However, IL4 has a downmodulating effect on memory T helper cell induction when IL2 is not produced. These results confirm that synergy between IL2 and IL4 is mandatory for the directive role of IL4 upon IL4-producing cells. Furthermore, the finding that IL4 promotes the induction of IFN gamma in a CSA-resistant pathway represents a new tool for analysis of regulation of the IFN gamma gene.

The J beta segment of the T cell receptor contributes to the V beta-specific T cell expansion caused by staphylococcal enterotoxin B and Urtica dioica superantigens.

We have used a new polymerase chain reaction-based technique to analyze at the clonal level the CDR3 diversity and the J beta usage associated with the V beta-dependent T cell receptor (TCR) recognition of two superantigens: the staphylococcal enterotoxin B and the Urtica dioica agglutinin. Our results show that subset of J beta elements is preferentially expanded in a given V beta family, independently of the nature of the superantigen. By contrast, the CDR3 loop does not contribute significantly to the T cell expansion induced by the superantigens. We conclude that the J beta segment of the TCR beta chain, but not the CDR3 region, participates in superantigen binding, presumably by influencing the quaternary structure of the TCR beta chain.

V beta-specific deletion of mature thymocytes induced by the plant superantigen Urtica dioica agglutinin.

Urtica dioica agglutinin (UDA), a plant protein, is a superantigen activating in a MHC class II-restricted manner the V beta 8. 3-bearing T-cells (Galelli and Truffa-Bachi, J. Immunol. 151, 1821, 1993). Administration of UDA to adult mice provokes the clonal expansion of the responding cells which is followed by the deletion of the major fraction of the UDA-sensitive cells, whereas the remaining cells become anergic (Galelli et al., J. Immunol. 154, 2600, 1995). We have analyzed the effect of UDA on thymocytes. Injection of UDA resulted in a rapid, but transient, deletion of a large fraction of the V beta 8.3-bearing mature T-cells. In contrast to other exogenous superantigens, this deletion was not preceded by the clonal expansion of the UDA-responding thymocytes. Moreover, the V beta 8.3-bearing mature T-cells escaping the deletion were not anergic to an in vitro UDA restimulation. UDA and the other superantigens also differ as the general, V beta-unrestricted, thymic atrophy induced by classical superantigens was not observed with UDA.

Gamma interferon and interleukin-10 gene expression in innately susceptible and resistant mice during the early phase of Salmonella typhimurium infection.

Previous studies have shown that gamma interferon (IFN-gamma) plays a major role in natural resistance to Salmonella typhimurium during the early phase of infection. To assess whether the level of natural resistance in mice is related to the level of IFN-gamma gene expression, we compared IFN-gamma mRNA levels by means of reverse transcriptase-PCR in the spleens of genetically susceptible Itys (C57BL/6 and BALB/c) and resistant Ityr (CBA and DBA/2) mice during the first 5 days of infection. The mRNA expression of interleukin-10 (IL-10), a cytokine which antagonizes IFN-gamma effects, was also investigated. Mice were infected with 10(3) CFU of the virulent strain S. typhimurium C5, a dose which is lethal within a week for susceptible mice only. IFN-gamma mRNA increased to similar levels in both susceptible and resistant mice, suggesting that susceptibility to S. typhimurium infection is not related to defective IFN-gamma gene expression. In contrast, IL-10 mRNA reached much higher levels in susceptible than in resistant mice. Similar results were found in Ity congenic mice, confirming a link between the presence of the Itys allele and a high level of IL-10 gene expression during infection. High levels of IL-10 mRNA in susceptible mice correlated with high IL-10 serum levels (on day 5), whereas IL-10 was not detectable in the sera of resistant mice. However, administration of neutralizing anti-IL-10 monoclonal antibodies did not modify the course of infection. To evaluate the influence of bacterial multiplication on IL-10 mRNA expression, susceptible mice were infected with an attenuated strain of S. typhimurium. This strain induced a low level of IL-10 mRNA expression. When susceptible mice were immunized with an attenuated strain and challenged with the virulent strain, they inhibited the growth of the challenge bacteria and exhibited a low level of IL-10 mRNA. In contrast, when resistant mice were infected with a high (lethal) dose of the virulent strain, they exhibited a high level of IL-10 mRNA. Taken together, these results indicate that the level of IL-10 gene expression correlates with the level of bacterial multiplication in the organs and that the high level of IL-10 mRNA in Itys mice is a consequence rather than the cause of their susceptibility to S. typhimurium infection.

Interleukin-2 down-modulates memory T helper lymphocyte development during antigenic stimulation in vitro.

Using an in vitro antigenic stimulation model of murine spleen cells in the presence of the immunosuppressor cyclosporin A (CSA) we have previously reported that not only does this drug not interfere with the differentiation of T lymphocytes into memory cells it appears to favor this differentiation (Motta, I. et al., Eur. J. Immunol. 1991. 21:551). Because CSA blocks interleukin-2 (IL-2) gene expression, we have analyzed the effect of this cytokine on memory T helper cell development. Murine splenic cells were primed for 6 days with sheep red blood cells (SRBC) in protocols in which either IL-2 was not produced or its biological activity was neutralized by anti-IL-2 receptor (R) antibodies. The helper function of the recovered T cells was revealed by their capacity to help virgin B splenocytes produce anti-SRBC antibodies upon challenge in vitro. We found that CD4+ cells primed in the absence of IL-2, provoked either by IL-2 gene transcription blockade by CSA or by treatment with anti-IL-2R antibodies, afford the best helper functions. These cells exhibit a memory-type phenotype characterized by the low expression of the MEL-14 marker and the high expression of the CD44 marker. Evidence is also presented that memory T helper cells originate in part from naive subset displaying the MEL-14hi phenotype. The pattern of expression of the genes encoding different cytokines (IL-2, IL-4, IL-5 and interferon-gamma) following a secondary antigenic stimulation shows that the helper function of the cells primed in the absence of IL-2 correlates with the up-regulation of the IL-2 and the IL-5 genes. From these data, we conclude that IL-2 plays a major role in the control of memory T helper cell induction.