IL-3-producing basophils are required to exacerbate airway hyperresponsiveness in a murine inflammatory model.

BACKGROUND: Basophils are commonly associated with allergic responses because of their ability to produce large amounts of pro-Th2 cytokines and histamine. However, the mechanisms through which bone marrow-resident basophils (BMRB) become fully competent cytokine and histamine producers in response to IgE crosslinking are poorly understood. Here, we sought to determine the role of IL-3 in promoting pro-Th2 basophils. METHODS: BMRB and basophils exposed to IL-3 in vitro and in vivo were evaluated for their production of Th2 cytokines and histamine in response to FcεRI crosslinking on both protein and gene expression levels. In vivo relevance of our findings was assessed in a model of ovalbumin-induced allergic asthma using IL-3-deficient and wild-type mice in a protocol of adoptive basophil transfer. RESULTS: We show that BMRB and basophils previously exposed to IL-3 differ in their ability to generate cytokines (IL-4, IL-6, IL-13, and GM-CSF) and histamine in response to FcεRI crosslinking, reflecting two stages of maturation. Exposure to IL-3 initiated an autocrine loop of endogenous IL-3 production that enhanced histamine and cytokine production upon FcεRI crosslinking. This increased responsiveness required calcium flux and was dependent on calcineurin and store-operated calcium channels. Our findings are of pathophysiological relevance, as assessed by the failure of IL-3-deficient mice to develop airway hyperreactivity, which could be restored by adoptive transfer of IL-3-derived basophils recovered from wild-type mice. CONCLUSION: IL-3-dependent basophils promote Th2 allergic AHR, which designates the IL-3/basophil axis as a promising therapeutic target for the treatment of basophil-dependent asthma.

Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation.

To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

Expression of functional major histocompatibility complex class II molecules on HMC-1 human mast cells.

Mast cells hold a key position in the defensive mechanisms against exogenous intruders. In this study, we investigated whether human mast cells express functional major histocompatibility complex (MHC) class II molecules that can transduce endogenous signals and present staphylococcal enterotoxin A (SEA) to T cells. Similar to HMC-1 human mast cell line, umbilical cord blood-derived mast cells express HLA-DR, -DP and -DQ molecules on their surface. MHC class II molecules expressed on HMC-1 cells bind significantly the SEA (a natural MHC class II ligand), and their ligation with specific mAbs or with SEA, leads ultrastructural changes, suggesting their degranulation. Recognition of SEA-bound MHC class II molecules on HMC-1 mast cells by the T cell receptor of K25 cells, an SEA-specific murine T cell hybridoma, triggers significant IL-2 secretion by these T cell hybridomas. Hence, our data point out the expression of functional MHC class II molecules on human mast cells, reinforcing the implication of these cells in the defense mechanisms of acquired immunity.

Anti-histone autoantibodies react specifically with the B cell surface.

In an attempt to induce an immune response against Mls-1a antigens by immunizing C57B1/6 mouse (Mls-1b) with purified B cells from DBA/2 mouse (Mls-1a), we generated a panel of monoclonal antibodies from which the 5B9.6 mAb, taken as a representative antibody, was thoroughly investigated. This antibody specifically reacts with B cells from all mouse strains studied including C57Bl/6 mice as shown by FACS analysis of double-antibody labelled spleen cells. Using enzyme immunoassays and immunoprecipitation techniques, 5B9.6 mAb was found to be specific for histones. Amino acid sequence analysis of a peptide derived from a 5B9.6-immunoprecipitated polypeptide from DBA/2 B cells showed a 100% homology with a sequence within H2B histones. Furthermore, 5B9.6 mAb was able to interact with the cell surface of 7OZ/3 cell line, known as a typical pre-B cell line. The presence of histones can be modulated on the surface of 7OZ/3 cells since this antigen was upregulated after exposure of these cells to a cocktail of IL-1 and cAMP. Finally, 5B9.6 mAb was shown to interact with freshly isolated B cells from human peripheral blood.

Inhibition of the passive cutaneous anaphylaxis reaction to Dactylis glomerata pollen allergens by a purified component of this pollen.

By isoelectric focusing a protein fraction of pI 4.6 was isolated from a crude water-soluble extract of Dactylis glomerata pollen (SE). This fraction was neither immunogenic nor allergenic in BALB/c mice. In one week, this protein inhibited the mouse IgE-specific antibodies to the soluble extract as measured by PCA in rats and was therefore called Dactylis inhibitory protein (DIP). Two experimental approaches which lowered IgE anti-SE titer were undertaken. Pretreatment with DIP as well as injection of DIP after the last sensitizing injection with SE resulted in an inhibition of the circulating IgE antibody level to SE. For both experiments the regulation of the immune response touched only the IgE class, whereas the titers of anti-SE IgG, IgM, IgA antibodies were not modified. DIP treatment did not alter the IgE titers, measured by PCA, in the immune response to ovalbumin.

In vivo presentation of Mls-1 antigen by T and B lymphocytes.

Previous studies of minor lymphocyte stimulatory (Mls) presenting lymphoid cells had shown that B cells rather than T cells present stimulatory Mls-1 antigen in vitro whereas B as well as T cells present Mls-1 antigen in vivo. Deletion of Mls-1 reactive T cells in the thymus of newborn mice is induced by T cells rather than by B cells. Applying a recently developed method for measuring the Mls-1 response in Mls-1- mice we assessed the Mls-1 stimulatory activity of T and B cells quantitatively. B cells are significantly more effective than T cells in this process. Both Mls-1+ T and B cells are also capable of inducing Mls-1 anergy in Mls-1- mice. Remarkably few lymphoid cells from Mls-1+ animals are needed for this effect: a few thousand B cells or 10(4) to 10(5) T cells per mouse induce substantial Mls-1 anergy in Mls-1- mice. These low cellular requirements for Mls-1 anergy production correspond well to the low T cell requirements described for the induction of Mls-1 tolerance in newborn mice. However, the high efficacy of B cells in inducing peripheral Mls-1 anergy contrasts with their failure to induce neonatal tolerance in newborn animals. We attribute this discrepancy to the previous notion that stimulatory Mls-1 antigen is not delivered to the thymus and that B cells and T cells present qualitatively different Mls-1 related signals to Mls-1 reactive T cells.

Differential expression of T cell receptor variable beta genes on CD4+ and CD8+ T cells: influence by sex linked genes?

We examined the expression of seven V alpha or V beta T cell receptor (TCR) segments on human CD4+ and CD8+ T cells. Confirming previously published results, we found a preferential expression of four V segment gene products on CD4+ T cells. One of these markers (V beta 6.7) was constantly expressed on more CD4+ T cells than CD8+ T cells. None of the analyzed blood samples showed a complete deletion of T cells expressing a particular V beta gene segment. In addition, our data provide the first evidence that genes on sex chromosomes may influence the formation of the human T cell repertoire. The ratio of CD4+/CD8+ T cells expressing V beta 12 gene products was always > or = 1 in female donors, whereas approximately 30% male donors exhibited more CD8+V beta 12+ T cells than CD4+V beta 12+ T cells.

[Immunological and non immunological mechanisms in urticaria]. / Mécanismes immunologiques et non immunologiques des urticaires.

Urticaria involve mast cell activation which could be mediated by immunological or non-immunological mechanisms. Interaction of allergens with the IgE/IgE receptor at the surface of mast cells has been postulated as the main immunologic type of mast cell activation. However, recent experimental and clinical studies have highlighted the existence of other mechanisms involving specific antibodies and T cells. IgG antibodies of different specificities (anti-IgE and/or anti-IgE receptor autoantibodies) have been characterized in a subgroup of patients suffering from chronic "autoimmune" urticaria. Circulating immune complexes may activate mast cells by interaction with the membrane-bound receptor for IgG. Interaction of mast cells with specific T cells could induce mast cell activation. Thus, immune-mediated urticaria appears to be secondary to different types of mast cell activation which could explain the various clinical presentation of the disease.

Comparison of the allergenic potency of spores and mycelium of Cladosporium.

The allergenic potency of spore and mycelium extracts of Cladosporium was estimated by RAST, RAST inhibition and PCA tests. Spores contained a concentration of allergens higher than mycelia. Results of PCA tests suggested that spores contained specific allergens. However, in a comparative study of extracts from different species of Cladosporium animal and human models gave different estimates of the allergenic potency of the different species. In spite of these variations it was shown that extracts from spores of Cladosporium contained the highest amount of Cladosporium allergens.

Purification and characterization of a major allergen from Dactylis glomerata pollen: the Ag Dg1.

We have isolated an allergen (Ag Dg1) from Dactylis glomerata pollen which is recognized by the serum of 95% of human patients sensitive to D. glomerata pollen, as has been shown by the nitrocellulose immunoprint technique. After two successive purifications by preparative isoelectric focusing (IEF), Dg1 was characterized as a single band in analytical agarose IEF with a pI of 5.9 and was found to display 3 bands by sodium dodecyl sulfate polyacrylamide gel with the respective molecular weights: 21,000, 31,000 and 33,000 daltons. The high recognition frequency by IgE antibodies of Dg1 in the sera of allergic patients and its ability to trigger histamine release from sensitized human basophils allow to consider that Ag Dg1 is the main major allergen extracted from D. glomerata pollen.

Induction of a passive cutaneous anaphylaxis inhibitory factor in mice.

The administration of a purified protein from Dactylis glomerata pollen, named Dactylis inhibitory protein (DIP), to Balb/C mice results in the production of a heat-resistant seric factor that blocks the passive cutaneous anaphylaxis (PCA) reaction in rat skin. This PCA inhibitory factor (PCA-IF) was purified following different purification procedures and analyzed in sodium dodecyl sulfate polyacrylamide gel. Amongst the array of bands detected it was possible to ascribe the inhibitory activity to a protein band of 76,000 daltons but only when it was associated either with another protein band of 69,000 daltons (obtained after a one-step purification procedure) or two other protein bands of 55,000 and 26,000 daltons (obtained after a three-step purification procedure). The inhibitory activity of the PCA-IF is present at a low level in normal Balb/C mice sera and is enhanced after DIP treatment of the mice.

B cells control the aggregability of CD4 on T cells through continuous physical interactions.

It has previously been demonstrated that a gene on chromosome 1 in or near Mls-1 controls, on the surface of B cells, the mobility and aggregability of major histocompatibility complex (MHC) class II molecules but not the mobility or aggregability of other B-cell molecules, such as immunoglobulin (Ig) and class I antigens. We report here that this gene may also influence the aggregability of two class II antigen-reactive molecules on the surface of T cells, the T-cell receptor complex and CD4. The aggregability of the two membrane components is markedly higher on Mls-1+ T cells than on Mls-1- T cells. The properties of this phenomenon were examined in vitro as well as in vivo with particular emphasis on CD4 aggregability. It was found that, after removal of B cells, T cells lose the ability to aggregate CD4 in our standard CD4 aggregation assay. Similarly, T cells isolated from the B-cell-deficient environment of the thymus failed to aggregate CD4. Addition of B cells to either thymic T cells or B-cell-depleted peripheral T cells established CD4 aggregability within minutes. This process can be blocked with antibody against CD4 or antibody against Ia. The Mls-1 genotype predicts within the limited tests of this study the efficacy of the B-cell ability to impose a CD4 aggregation pattern on T cells: Mls-1+ B cells are markedly more effective in this respect than Mls-1- B cells. This can be demonstrated in tissue culture as well as in the animal. Similar to the Mls-1 response, this is a one-way process: Mls-1+ B cells confer to Mls-1- mice a CD4 aggregation pattern typical of the Mls-1+ mouse while Mls-1- B cells do not impose a Mls-1b-typical CD4 aggregation pattern in Mls-1a mice. Mls-1+ B cells also influence the composition of lymphocytes in the mouse. Mls-1+ mice or Mls-1- mice treated with Mls-1+ B cells have fewer T cells and more B cells in their spleen than Mls-1- animals. The gene that encodes stimulatory Mls-1 cell-surface structures has recently been identified as an endogenous mammary tumour virus (Mtv-7). We expect that the analysis of the virus genome will produce information whether the effects described here can be attributed to the virus or not.

Induction of neonatal tolerance to the Mls-1a self-super-antigen. Time kinetics and MHC restriction.

We examined the accessibility of the thymus to a self-super-Ag encoded by the Mls-1a region of chromosome 1 and the process by which this Ag establishes immunologic tolerance. Intravenously administered Mls-1a Ag accumulates quickly in peripheral organs of adult or newborn Mls-1a- recipients, where it mounts an immune response. The Ag does not enter the thymus in detectable amounts and does not induce an immune response of Mls-1a-responsive T cells present in this organ. Instead, the thymus of newborn Mls-1a- recipients of Mls-1a+ lymphoid cells continues for several days to export Mls-1a-reactive T cells, which respond to Mls-1a Ag when they encounter it in peripheral organs. This response peaks around day 3 or day 4 and declines very rapidly thereafter. The deletion of intrathymic Mls-1a-reactive T cells ensues simultaneously with this decline. It has previously been shown that Mls-1a Ag causes deletion or anergy of Mls-1a-reactive peripheral T cells, subsequent to their activation. We see the same time kinetics in producing deletion or anergy of Mls-1a-reactive T cells in the thymus of newborn animals, with the exception that the activation phase that precedes the deletion of Mls-1a-reactive T cells occurs in the periphery and not in the thymus. This observation indicates that thymic Mls-1a-specific T cells are not deleted through activation. Whether their deletion depends on a feed-back from the peripheral activation of Mls-1a-reactive cells, as the time relationship could suggest, is not clear. The finding establishes, however, that the deletion of functionally mature Mls-1a-reactive T cells and the activation of such cells are not necessarily related events, which may or may not utilize a common trigger mechanism, such as the engagement of the TCR. Concerning the trigger mechanism, we report that Mls-1a-specific deletion of T cells is an MHC-restricted process, whereas Mls-1a-specific activation of T cells is not MHC restricted.

Regulation of T cell function by Mtv-7 gene products.

Mls-1, a superantigen encoded by the endogenous mouse mammary tumor virus Mtv-7 induces immunological tolerance through deletion of antigen-reactive T cells. A remarkable difference between this self-antigen and self-MHC antigens is that while the mouse establishes tolerance against self MHC antigens by the time of birth it does not begin to delete T cells specific for the self-superantigen until they had mounted an immuneresponse against it. An immune response occurs normally several days after birth and may be delayed experimentally for weeks before the deletion process ensues. However, for effective deletion of Mls-1 reactive T cells the mouse must be exposed to Mtv-7 positive lymphoid cells within hours after birth. In reviewing here data obtained in this and other laboratories regarding experimental induction of Mls-1 tolerance in neonatal mice we are trying to make a case for the involvement of Mtv-7 encoded antigens distinct from the superantigen. We propose that T cells reactive with non superantigenic Mtv-7 determinants pose a threat to the establishment of chimaerism between Mls-1- neonates and Mls-1+ inocula, as they may cause the rejection of Mls-1 superantigen bearing lymphocytes. Chimaerism is essential for the establishment of lasting Mls-1 tolerance.

Biological effect of transferrin on mast cell mediator release during the passive cutaneous anaphylaxis reaction: a possible inhibition mechanism involving iron.

A purification procedure for passive cutaneous anaphylaxis inhibitory factor from BALB/c mouse serum has been previously described. In the present work, this inhibitory activity was found to be related to transferrin. No activity was obtained using iron-unsaturated transferrin, whereas iron-saturated transferrin appeared to be potent. The in vivo inhibition of IgE-dependent mediator release from rat mast cells was also obtained using free iron. This effect was observed when iron was injected prior to or simultaneously with mouse IgE in rat skin. Iron and iron-saturated transferrin could play a role in the mechanism of desensitization by modulating the responsiveness of mast cells.

Immunogenic peptides require an undisturbed phospholipid cell membrane environment and must be amphipathic to immobilize Ia on B cells.

Ia-reactive immunogenic peptides have been shown to immobilize Ia molecules on the B cell surface and to facilitate their aggregation with specific alloantibody. We show that to immobilize Ia the peptide must be amphipathic. Polar peptides appear to bind to Ia molecules as judged by competitive inhibition, but do not immobilize the MHC molecule. This suggests the possibility that peptides establish the immobilizing membrane contact via a lipophilic group. Examining the B cell membrane lipid environment, we found that treatment of B cells with phospholipase C prevents peptide-mediated immobilization of Ia. The requirement of a lipophilic peptide portion as well as of phospholipase-sensitive membrane components for effective peptide-mediated Ia aggregation on B cell membranes suggests a role for membrane phospholipids in this process. We advance the speculation that immunodominant amphipathic peptides immobilize Ia molecules by attaching them to cell surface phospholipids which we tentatively refer to as immobilizing phospholipids.

Production of a monoclonal antibody against a major allergen of Dactylis glomerata pollen (Dg1).

A mouse monoclonal antibody was produced against the most frequent allergen Ag Dg1ief, purified from the Dactylis glomerata grass pollen by isoelectric focusing. Using the immunoprint technique, this monoclonal antibody detected Ag Dg1ief in D. glomerata pollen and some cross-reacting molecules in four other grass pollens. This hybridoma was grown in ascitic fluid. An immunosorbent was made from it and, by affinity chromatography, was used to purify monoclonal Ag Dg1m from the pollen extract. Ag Dg1m (6,000 to 8,000 daltons) appeared to be 4 to 5 times smaller than Ag Dg1ief. It appeared as a weak allergen in passive cutaneous anaphylaxis in rats, but was more potent in triggering histamine release on human basophils from grass-pollen sensitive patients. These results suggest that Ag Dg1m could be a part of the Ag Dg1ief used to induce production of this monoclonal antibody.

Interleukin-2 may enhance or inhibit antibody production by B cells depending on intracellular cAMP concentrations.

The primary IgM response of murine B lymphocytes against red blood cell-bound antigens can be induced by incubating antigen-reactive B cells either with the lymphokines interleukin-1 (IL-1) and IL-2 together with the nucleoside cAMP, or by the addition of antigen-specific helper T cells. The reactivity of B cells is strongly influenced by the T-cell lymphokine IL-2. IL-2 inhibits the cyclic adenosine 3',5'-phosphate (cAMP)-dependent B-cell response when it is allowed to act on the cells prior to cAMP. On the other hand, if IL-2 acts on B cells together with or after cAMP, it synergizes with the nucleoside and enhances the immune response. A similar effect of IL-2 is observed in the T-cell-mediated activation of B cells. If IL-2 is present before helper T cells interacted with B cells, it inhibits antibody production. The inhibitory IL-2 effect is reversed by the simultaneous addition of exogenous cAMP. The finding supports the hypothesis that Ia ligation by T cells results in B cells in the elevation of cAMP which acts as an important second messenger in B cells. The antagonism between cAMP and IL-2 was also examined in the pre-B-cell line 70Z/3. The nucleoside is highly toxic to 70Z/3 pre-B cells and a majority disintegrates within hours of exposure to the nucleoside. The surviving cells undergo phenotypic differentiation expressing surface Ig kappa chains and major histocompatibility complex (MHC) class II molecules, and increase the expression of IL-2 receptor (R). The phenotypic differentiation requires the presence of IL-1. IL-2 inhibits both of these B-cell responses to cAMP, the IL-1-independent cell death, and the IL-1-dependent phenotypic differentiation.

A stimulatory Mls-1 superantigen is destroyed by ultraviolet light while other Mtv-7 antigens remain intact. Significance for Mls-1 unresponsiveness.

Accessory cells present Ag together with costimulatory signals as immunogens and without costimulatory signals as tolerogens. Responsiveness and unresponsiveness are thus alternatives of T cell immune reactions to Ag. Superantigens appear to make an exception; being presented by accessory cells capable of providing costimulatory signals, these Ag induce a strong T cell response but leave T cells unresponsive to a secondary challenge (anergy). We show here that T cell anergy is not a mandatory consequence of superantigen-induced activation. Mls-1- BALB/c recipients of DBA/2 spleen cells mount vigorous Mls-1 responses in vivo but their T cells retain the ability to respond to a subsequent Mls-1 challenge in vitro. We tested the possibility that the inability of DBA/2 spleen cells to inactivate Mls-1-reactive BALB/c T cells was the result of excessive costimulatory activity provided by Mls-1+ DBA/2 B cells. Costimulatory accessory cell activity has been reported to be destroyed by UV light. We exposed superantigen-presenting cells to UV radiation and found that they had lost the ability to stimulate an Mls-1 response without, however, gaining the capacity to render Mls-1-specific T cells anergic. Despite their inability to noticeably stimulate Mls-1-reactive T cells, UV-treated Mls-1+ lymphocytes induced an absolute unresponsiveness in Mls-1- recipients to a second challenge with the superantigen. Our data are in agreement with previous evidence, confirmed here, that BALB/c mice establish immunity against Mls-1+ cells, which causes the accelerated rejection of superantigen-bearing lymphocytes. Thus, our data imply that, whereas it takes stimulatory superantigenic Mtv-7 gene products to induce the activation of superantigen-reactive T cells, nonsuperantigenic Mtv-7 gene products may induce an immune response leading to the elimination of Mtv-7+ lymphoid cells.