SumlTlaryAsthma is a chronic life-threatening disease of worldwide importance. Although allergic asthma and related atopic conditions correlate strongly with immune sensitization to house dust mites, it is unclear why antigens from mites provoke such powerful allergic immune responses. We have characterized the protease activity of Der p I, the group I protease allergen of the house dust mite Dermatophagoides pteronyssinus, and here report that it cleaves the low-affinity immunoglobulin (Ig) E Fc receptor (CD23) from the surface of human B lymphocytes. Derp I selectively cleaves CD23 and has no effect on the expression of any other B cell surface molecules tested. We speculate that this loss of cell surface CD23 from IgE-secreting B cells may promote and enhance IgE immune responses by ablating an important feedback inhibitory mechanism that normally limits IgE synthesis. Furthermore, since soluble CD23 is reported to promote IgE production, fragments of CD23 released by Der p I may directly enhance the synthesis of IgE. cel-Antiprotease, a pulmonary antiprotease, is also shown to inhibit the cleavage of CD23 by Derp I. This may be significant in the etiopathogenesis of asthma, because other indoor pollutants associated with asthma are known to potently inhibit this antiprotease. These data suggest that the proteolytic activity of Derp I, the group I allergen of the house dust mite D. pteronyssinus, is mechanistically linked to the potent allergenicity of house dust mites. Furthermore, inhibition of Der p I by cq-antiprotease suggests a mechanism by which confounding factors, such as tobacco smoke, may act as a risk factor for allergic asthma.

Previously, we had shown that T cells accumulated in peribronchiolar and perivascular areas of lungs soon after intranasal infection with Streptococcus pneumoniae. We have now presented new evidence, using major histocompatibility class II-deficient mice, that CD4 cells are important for early protective immunity. In addition, we have also shown that a population of human CD4 cells migrates towards pneumococci and that in vivo-passaged pneumococci are substantially more potent at inducing migration than in vitro-grown bacteria. This migratory process is unique to a specific population of CD4 cells, is highly reproducible, and is independent of prior CD4 cell activation, and yet the migratory process results in a significant proportion of CD4 cells becoming activated. The production of pneumolysin is a key facet in the induction of migration of CD4 cells by in vivo bacteria, as pneumolysin-deficient bacteria do not induce migration, but the data also show that pneumolysin alone is not sufficient to explain the enhanced migration. Increased CD25 expression occurs during migration, and a higher percentage of cells in the migrated population express gamma interferon or interleukin 4 (IL-4) than in the population that did not migrate. There is evidence that the activation of IL-4 expression occurs during migration.

The proteolytic activities frequently associated with sources of allergens and parasite secretions have been suggested as important immunomodulators. We have investigated whether the protease activity of the house dust mite allergen Der p1 and the secreted proteases of the hookworm Necator americanus are able to directly induce type 2 cytokine production by basophils. Der p1 and the secretions of N. americanus induced interleukin (IL)-4, IL-5, and IL-13 but not interferon-gamma mRNA in KU812 basophils. Enzyme-linked immunosorbent assay confirmed that IL-4 and IL-13 were secreted. A nonproteolytic antigen failed to induce cytokine expression, and preincubation of Der p1 or N. americanus secretions with protease inhibitors inhibited cytokine expression. Data were confirmed using basophils purified from human peripheral blood. We speculate that this innate mechanism may contribute to the development of a cytokine milieu that could promote immunoglobulin E synthesis, eosinophil recruitment, and the development of type 2 T cells.

SummaryThe Staphylococcal enterotoxin superantigens stimulate vigorous responses in T cells bearing certain T cell antigen receptor (TCR) V~ regions. In addition to activation, these superantigens also impart negative signals to T cells resulting in a profound state of unresponsiveness or anergy. The Staphylococcus aureus enterotoxins (SE) B and C2 bind to a closely related site on major histocompatibility complex (MHC) human leukocyte antigen (HLA)-DK1 molecules. Only SEB, however, interacts with the TCR V/33 region of HA1.7, a human HLA-DK1 restricted T cell done specific for influenza haemagglutinin. In competition experiments, we demonstrated that the induction of anergy in HA1.7 by SEB is unaffected by the presence of SEC2. These results suggest that SEB-induced anergy is MHC independent and involves a direct interaction between the TCR and SEB. To resolve definitively whether SEB binds directly to T cells in the absence of MHC class II molecules, the cDNAs encoding the HA1.7 TCK were transfected into an MHC class II-negative human T cell line. The addition of SEB to these transfectants resulted in the downregnlation of cell surface TCK expression, an increase in the concentration of intracellular calcium ions, the production of lympholcines, and reduced responsiveness to a subsequent challenge with SEB. We conclude that SEB interacts directly with the TCR in the absence of cointeraction with MHC class II molecules, and that this interaction may induce anergy in HA1.7. group of exotoxins produced by certain strains of Staphylococcus aureus induce vigorous responses in T cells expressing particular TCR VB elements (1-4). Such exotoxins have thus been termed superantigens. In common with endogenous murine retroviral superantigens, bacterial superantigens also have the capacity to shape the TCR repertoire by clonal deletion (2, 5) or the induction of anergy (6-8). Since thymic deletion alone fails to explain self-tolerance to antigens expressed exclusively by adults or in sites remote from the neonatal thymus, anergy, which can be induced in mature T cells, is proposed to account for T cell unresponsiveness to these antigens both in in vivo (6-9) and in vitro models (10-12).Incubation of human T cell clones with supraoptimal concentrations of nominal peptide antigen renders the cells anergic to a subsequent immunogenic challenge (10,11,13). In common with antigen-specific T cell activation, antigeninduced anergy is initiated by MHC class II-restricted antigen presentation (14).More recently, it has been demonstrated that Staf~lococcus aureus enterotoxin (SE) 1 superantigens, are also able to induce T cell anergy to their native ligand (12,15). Unlike peptide antigen-induced anergy, however, enterotoxin-induced anergy was not inhibited by anti-MHC class II mAbs (R. E. O'Hehir and J. K. Lamb, unpublished observations).The aim of the present study was, therefore, to determine whether a Staphylococcal enterotoxin superantigen was able to interact directly with the TCK and induce donal anergy in the absence of MHC-...

Eosinophil recruitment to airway tissue is a key feature of asthma, and release of a wide variety of toxic mediators from eosinophils leads to the tissue damage that is a hallmark of asthma pathology. Factors that control the release of these toxic mediators are targets for potential therapeutic intervention. Protease-activated receptors (PARs) are a novel class of receptors that are activated by cleavage of the N terminus of the receptor by proteases such as thrombin or trypsin-like enzymes. To date, PAR1-4 have been identified, and there are several studies that have demonstrated the expression of PARs in airway tissue, particularly the respiratory epithelium. We have investigated whether eosinophils express PARs and if activation of these receptors will then trigger a functional response. Using a combination of reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometry analysis, we have demonstrated that eosinophils express PAR1 and PAR2. FACS analysis showed that PAR1 could be clearly detected on the surface of the cells, whereas PAR2 appeared to be primarily intracellular. Trypsin and the PAR2 agonist peptide were seen in trigger shape change, release of cysteinyl leukotrienes, and most obviously, generation of reactive oxygen species. In contrast, thrombin had no effect on eosinophil function. The PAR1 agonist peptide did have a minor effect on eosinophil function, but this was most likely down to its ability to activate PAR1 and PAR2. These results demonstrate that PAR2 is the major PAR receptor that is capable of modulating eosinophil function.

Sensitivity to house dust mite antigens in atopic individuals is a major cause of allergic diseases, ranging from asthma to rhinitis and dermatitis. We have studied the T-cell receptor (TCR) usage of house-dust-mite-specific CD4+ T-cell clones isolated from an atopic individual, by using the anchored polymerase chain reaction, and have analyzed the peripheral TCR repertoire of the same individual. Several T-cell clones had identical TCRs at the sequence level, despite the fact that they had been independently isolated, in some cases, in different years. These data suggest the presence in vivo of long-lived T-cell clones. We have also shown that junctional sequences identical to these clones are present in peripheral blood T cells taken 6 years after the isolation of the T-ceil clones.

Immmoglobulin E-rich plasma from patients from Papua New Guinea infected with Necator americanus has been used to probe an adult N. americanus cDNA library for the presence of hookworm allergens. Using this approach, one hookworm allergen has been identified as calreticulin, which was subsequently expressed in Escherichia coli. Little serological cross reactivity was seen between the recombinant calreticulins of this hookworm and its host. Prospective roles for hookworm calreticulin in the host-parasite relationship are discussed in depth.

Superantigens are able to stimulate T lymphocyte populations expressing T cell antigen receptors (TCR) belonging to particular V beta families. Moreover, the presence of these superantigens may induce long term unresponsiveness (anergy) of these sensitive cells. Some bacterial toxins are potent superantigens. We have analyzed in vitro the capacity of some Staphylococcus aureus enterotoxin superantigens to modulate T cell antigen receptor expression and the cellular mechanisms involved. Staphylococcus enterotoxin B (SEB) induced rapid down-regulation of surface T cell antigen receptors in V beta 3-expressing T lymphocytes, as assessed by flow cytometry. This phenomenon was a consequence of the direct interaction between the toxin and the TCR since it was observed in the absence of cells expressing major histocompatibility complex class II molecules. The cellular mechanism involved in SEB-induced down-regulation of TCR was further investigated. Immunofluorescence and confocal microscopy experiments showed that toxin B induced intracellular accumulation of TCR.CD3 in endocytic vesicles. Moreover, SEB induced an increase in T cell receptor endocytosis as measured using radiolabeled Fab fragments of an anti-CD3 monoclonal antibody. Taken together, our observations indicate that Staphylococcus enterotoxin B superantigen induced changes in the dynamics of surface T cell receptors, which resulted in the fast reduction of membrane receptor numbers.