CD23 and allergic pulmonary inflammation: potential role as an inhibitor.

CD23, a receptor for immunoglobulin E, is expressed at increased levels in asthmatic and atopic individuals and has been associated with disorders characterized by chronic inflammation. Using an established murine model, we employed several complementary strategies to investigate the role of CD23 in allergic pulmonary inflammation and airway hyperresponsiveness (AHR). Specifically, these approaches included the modulation of CD23 function in vivo by administration of anti-CD23 monoclonal antibody (mAb) or Fab fragments to wild-type mice and the analysis of CD23-deficient mice. Administration of anti-CD23 mAb, but not anti-CD23 Fab fragments, produced attenuation of pulmonary inflammation, AHR, and CD8(+) T-cell activation. On the basis of a model that the anti-CD23 mAb transduces, whereas the Fab fragment inhibits, CD23 signaling, these results suggest that CD23 negatively regulates pulmonary inflammation and AHR. This hypothesis is supported by our observation that CD23-deficient mice developed increased inflammation and AHR after sensitization and challenge with allergen. Together, these results indicate that CD23 negatively regulates pulmonary inflammation and airway hyperreactivity.

Detection of rare apoptotic T cells in vivo.

The flow cytometric analysis of apoptosis in lymphocytes from in vivo samples has been difficult because of the low frequency of apoptotic events. To overcome this obstacle, many investigators have relied on in vitro incubations to increase the number of apoptotic cells before analysis. In this report, we show that an adaptation of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay for use in flow cytometry can be used to detect rare apoptotic lymphocytes from freshly harvested LN suspensions. This approach is both specific and extremely sensitive. This method also is amenable to multiparameter analyses and allows a phenotypic analysis of these rare apoptotic cells. However, we observed that some monoclonal antibodies can stain apoptotic-but not viable-cells nonspecifically. Therefore, the specificity of all antibodies to stain apoptotic cells was confirmed in competition assays.

Fas modulation of apoptosis during negative selection of thymocytes.

A major mechanism maintaining immune tolerance is the deletion of potentially autoreactive thymocytes by apoptosis during development in the thymus. Previous reports suggest that apoptosis is induced by high avidity signals transduced via the T cell receptor; however, the role of signals transduced by other cell surface receptors during thymic selection remains poorly understood. Fas, a member of the TNF receptor family, has been shown to induce apoptosis in mature peripheral T cells; however, the effects of Fas on negative selection of thymocytes have not been previously detected. Using a sensitive terminal deoxynucleotidyl transferase method to detect apoptotic cells, we found that mutant Fas molecules in lpr mice decrease the sensitivity of thymocytes to T cell receptor-mediated apoptosis and that blockade of Fas-Fas ligand interactions in vivo can inhibit antigen-induced apoptosis of thymocytes in non-lpr mice. Thus, we have shown that Fas, in conjunction with antigen-specific signals, can modulate apoptosis during negative selection of thymocytes.

T cell activation in a murine model of asthma.

To determine the mechanisms by which inhaled antigens produce pulmonary inflammation and bronchial hyperreactivity, we have developed a murine model of asthma. BALB/c mice are sensitized and challenged with ovalbumin (OVA). Compared with mice treated with phosphate-buffered saline (PBS), OVA-treated mice developed increased lung resistance, decreased dynamic compliance, and greater methacholine reactivity. Bronchoalveolar lavage fluid revealed significant increases in the proportion of neutrophils and eosinophils. Tissue sections of OVA-treated mice demonstrated goblet cell metaplasia and focal perivascular and peribronchial infiltrates composed of lymphocytes, neutrophils, and eosinophils. Analysis of thoracic lymphocytes via flow cytometry revealed an expansion of both CD4+ and B cell populations, with increased expression of interleukin-2 receptor on CD4+ T cells, indicated increased activation. There was also increased expression of CD44 on CD4+ and CD8+ lymphocytes, suggesting an expansion of the local memory cell population. These findings support the hypothesis that activation of T lymphocytes mediates allergic pulmonary inflammation and bronchial reactivity in asthma.

Restriction of the TCR repertoire inhibits the development of memory T cells and prevents autoimmunity in lpr mice.

The lpr mutation, a disruption of the fas gene, induces spontaneous autoimmunity characterized by high titers of autoantibodies, lymphadenopathy, autoreactive T cells, and early mortality. The mechanism of autoimmunity, however, remains unknown. The driving force for disease could result from the T cell recognition of autoantigen or, alternatively, an intrinsic T cell defect that promotes autoreactivity. We investigated the role of antigen-TCR interaction in the pathogenesis of lpr autoimmunity by transferring the DO-11.10 TCR beta-chain transgene (Vbeta8.2-Dbeta1.1-Jbeta1.1) to the MRL-lpr/lpr background producing the MRL-lprbeta strain. Our results show that the MRL-lpr beta transgenic strain has increased survival, lower titers of autoantibodies, and decreased lymphadenopathy compared with nontransgenic littermates. These beneficial effects were associated with decreased expansion of CD4+ T cells expressing memory phenotypes (CD44+, CD45RB-, and LECAM-) in the transgenic compared with nontransgenic strains. A role for impaired recognition of autoantigen by T cells expressing the TCR transgene was suggested by comparing the phenotypes of Vbeta8.2+ (transgene+) vs Vbeta8.2- (transgene-) CD4+ T cells within the transgenic mice. These experiments show that Vbeta8.2- T cells, which express endogenously rearranged TCR, are the major contributors to the expansion of memory T cells in the transgenic mice. In contrast, T cells with memory phenotypes expand similarly in both the Vbeta8.2+ and Vbeta8.2- subsets of nontransgenic mice. Based on these results, we hypothesize that TCR recognition of autoantigen is a major contributor to autoimmunity in lpr mice and that T cells expressing a memory phenotype are perpetrators of this process.

Plasticity of the T cell receptor repertoire in TCR beta-chain transgenic mice.

The potential alpha beta T cell receptor (TCR) repertoire in normal mice is extremely large and estimated by M. M. Davis and P. J. Bjorkman (Nature 334, 395, 1988) to include 5.2 x 10(18) different receptor molecules. This tremendous diversity provides the basis for T cell recognition of the universe of antigens including bacterial, viral, and allogeneic epitopes. Expression of a single TCR beta-chain transgene should alter the repertoire by limiting the available diversity, therefore, creating holes in the repertoire or producing TCR with decreased affinity. To determine the effect of drastically decreasing the size of the repertoire, we investigated T cell responses in TCR beta-chain transgenic mice expressing the V beta 8.2 transgene. Previous results showed that > 98% of T cells in these mice express the transgene; thus, the TCR repertoire is reduced by orders of magnitude. We tested the T cell responses of the transgenic mice and nontransgenic littermates to nine different MHC haplotypes in mixed lymphocyte reactions, five protein antigens, and eight immunogenic peptides. Surprisingly, the transgenic mice responded to all antigenic stimuli tested indicating the lack of a hole in the TCR repertoire. Interestingly, however, the response in every case was quantitatively lower than the response by the nontransgenic littermates. In contrast, transgenic and nontransgenic T cells responded equivalently to stimulation with mitogens or to stimulation with immobilized alpha-TCR mAb indicating that the transgenic T cells had a normal capacity to respond. To differentiate between decreased TCR affinity and decreased precursor frequency, we performed a limiting dilution analysis to the peptide antigens CI:NP and OVA324-339. The results showed approximately a three-to eight-fold decrease in the frequency of transgenic T cells responding to the peptide compared to nontransgenic littermates. We previously showed that the response to cI84-98 and PLP could be blocked with anti-V beta 8 mAb indicating that V beta 8.2-bearing T cells are capable of responding to peptide antigen. Analysis of TCR V alpha chain expression by PCR and flow cytometry showed similar V alpha expression in both the transgenic and the nontransgenic mice. These results demonstrate tremendous plasticity in the TCR repertoire permitting T cell responses by the transgenic mice to all antigens tested. However, the decreased magnitude of the responses may impair the capacity to defend against natural pathogens. Therefore, although the large TCR repertoire of normal mice may not be necessary to produce in vitro responses to many experimental antigens, it may confer survival benefits in natural environments.

Differential expression of activation markers during tolerance induction by superantigens in T-cell receptor (beta-chain) transgenic mice.

To investigate the process of tolerance induction we have developed an in vivo model using TCR beta-chain transgenic mice tolerized with the superantigen staphylococcal enterotoxin B. We have previously demonstrated that tolerized peripheral T cells were anergic when stimulated in vitro with immunogenic peptides, superantigens, mitogens, and immobilized anti-TCR mAb. However, the development of anergy is preceded by an induction phase which produces expansion followed by contraction of the peripheral T cell population presumably due to proliferation and programmed cell death, respectively. The current experiments focus on the induction phase of tolerance. A kinetic functional analysis showed that the inhibition of proliferation was apparent 2-3 days post-tolerization. Interestingly, the inhibition of proliferation correlated with the loss of IL-2R alpha expression, which occurred 2 days post-tolerization following an initial increase in IL-2R alpha expression. In addition, the expression of multiple activation markers including CD44, Ly-6A/E, and very early activation marker H1.2F3 is induced, whereas the expression of CD45RB is decreased during tolerance induction. Elevated expression of Ly-6A/E persists up to 28 days post-tolerization; however, altered expression of the other markers does not persist and near baseline levels of the other markers are noted 7 to 28 days post-tolerization. These results show that tolerance induction is an active process which has functional and phenotypic similarities to antigen-specific immunity. However, tolerance induction in our system differs from immunity in terms of the early loss of IL-2R alpha expression, the persistent increased expression of Ly-6A/E, and the lack of development of CD45RBlo memory-type T cells.