CD40L, but not CD40, is required for allergen-induced bronchial hyperresponsiveness in mice.

Asthma is characterized by immunoglobulin (Ig) E production, infiltration of the respiratory mucosa by eosinophils (EOSs) and mononuclear cells, and bronchial hyperresponsiveness (BHR). Interaction of CD40 on B cells and antigen presenting cells, with its ligand (CD40L) expressed transiently on activated T cells, is known to augment both T cell-driven inflammation and humoral immune responses, especially IgE production. Considering both the prominent role of inflammation in asthma and the association of the disease with IgE, we hypothesized that CD40-CD40L interactions would be important in pathogenesis. To test this hypothesis, we subjected wild-type (WT) mice and animals lacking either CD40 or CD40L to repeated inhalation of Aspergillus fumigatus (Af ) antigen. Af-treated WT mice displayed elevated IgE levels, bronchoalveolar lavage and pulmonary tissue eosinophilic inflammation, and BHR. IgE production was markedly suppressed in both the CD40 -/- and CD40L -/- strains. However, pulmonary inflammation did not appear to be inhibited by either of these mutations. Paradoxically, development of BHR was prevented by the lack of CD40L but not by the absence of CD40. We conclude that CD40/CD40L interactions, although critical in the induction of IgE responses to inhaled allergen, are not required for the induction of EOS-predominant inflammation. CD40L, but not CD40, is necessary for the development of allergen-induced BHR.

Genetic variability in pulmonary physiological, cellular, and antibody responses to antigen in mice.

Wide differences among inbred mouse strains in susceptibility to develop components of asthmalike pulmonary changes would provide insights into the nature of the relationships among those components and set the stage for genetic approaches to their etiology. We therefore examined pulmonary pathophysiological and serum immunoglobulin (Ig)E responses in mice of 12 inbred strains sensitized intraperitoneally with ovalbumin (OVA) and repeatedly exposed to aerosolized OVA. One day after the last OVA exposure the intravenous methacholine (MCh) dose required to reduce lung conductance by 50% (ED(50)GL) in OVA-sensitized and exposed mice was reduced by 0 to 2.7-fold, compared with sham-sensitized mice, depending on the strain. In OVA-sensitized mice, bronchoalveolar lavage (BAL) eosinophils comprised from 3.3 +/- 3.1 (SD) to 91.2 +/- 5.0% of BAL cells and eosinophilic pulmonary inflammation varied from being nondetectable to widespread and severe. OVA-specific IgE concentrations ranged from less than 3 ng/ml to 455 ng/ml in different strains. Shifts in responsiveness correlated significantly with pulmonary eosinophilia among strains (r > 0.70, p < 0.001) but not with antigen-specific IgE levels (r = 0.55, p = 0.056). These results demonstrate that allergen- induced enhancement of cholinergic responsiveness, pulmonary eosinophil influx, and elevations of serum antigen-specific IgE levels are each genetically determined and are not always associated.

Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus.

The Ig heavy chain (IgH) constant region (CH) genes are organized from 5' to 3' in the order Cmicro, Cdelta, Cgamma3, Cgamma1, Cgamma2b, Cgamma2a, Cepsilon, and Calpha. Expression of CH genes downstream of Cdelta involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding CH gene. Previously, we demonstrated that insertion of a PGK-neor cassette at two sites downstream of Calpha inhibits, in cultured B cells, GT of and CSR to a subset of CH genes (including Cgamma3, Cgamma2a, Cgamma2b, and Cepsilon) that lie as far as 120 kb upstream. Here we show that insertion of the PGK-neor cassette in place of sequences in the Igamma2b locus inhibits GT of and CSR to the upstream Cgamma3 gene, but has no major effect on the downstream Cgamma2a and Cepsilon genes. Moreover, replacement of the Cepsilon exons with a PGK-neor cassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream Cgamma3, Cgamma2b, and Cgamma2a genes. As with the PGK-neor insertions 3' of Calpha studied previously, the Cgamma1 and Calpha genes were less affected by these mutations both in culture and in mice, whereas the Cgamma2b gene appeared less affected in vivo. Our findings support the existence of a long-range 3' IgH regulatory region required for GT of and CSR to multiple CH genes and suggest that PGK-neor cassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent CH genes upstream of the insertion.

The expression of murine B cell CD23, in vivo, is regulated by its ligand, IgE.

CD23, the low-affinity IgE receptor, is believed to participate in immune responses by mediating antigen capture for presentation by B cells and by shedding fragments with immunomodulatory properties. The number of CD23 molecules on B cells is increased during allergic responses and infection with helminths. This can be attributed in part to regulation of CD23 expression by cytokines, including IL-4. In addition, there is evidence that CD23 can be induced on cultured B cells by its ligand, IgE. In the current study we use IgE-deficient (IgE-/-) mice to establish the effects of IgE on CD23 expression by B cells in vivo, in the absence of allergic or parasitic stimuli. The spleens of IgE-/- and wild-type mice contained similar proportions of CD23+ B lymphocytes. However, cells from IgE-/- mice were found to have nearly 3-fold less CD23 on their surface. The mutant B cells had a corresponding defect in their ability to bind IgE. CD23 could be normally induced on IgE-/- B cells after culture with IL-4 or CD40 ligand, indicating that these cells had no inherent defect in CD23 biosynthesis. CD23 expression and IgE-binding capacity were both restored when splenocytes from IgE-/- mice were cultured in the presence of IgE. IgE-induced up-regulation of CD23 could be elicited in vivo as well. In IgE-/- mice, i.v. infusion of IgE corrected CD23 expression to wild-type levels. Our results demonstrate that IgE directly participates in CD23 regulation in vivo. This positive feedback loop may constitute a mechanism for the amplification of ongoing allergic responses.