To our knowledge, this is the first demonstration of intact antigen-specific Th2 immunity upon mucosal sensitization of LTα-KO mice. Indeed, compared with WT mice, LTα-KO mice responded to OVA aerosolization in the context of GM-CSF with a similar pattern of T1/ST2 expression on Th2 cells and a robust Th2-associated inflammatory response characterized primarily by eosinophilia in the lung tissue. Moreover, after resolution of the initial response, OVA reexposure readily reconstituted airway eosinophilic inflammation, indicating antigen specificity and the persistence of immunological memory. As further evidence of Th2 immunity, LTα-KO mice were able to generate OVA-specific IgE responses. This is of particular interest in the light of previous data indicating the compromised ability of LTα-KO mice to generate antibody responses against KLH, HSV-1, and SRBC (9
). It is also of interest because germinal centers, which are considered to be essential for Ig isotype switching, are absent in these mutant mice. However, Matsumoto et al. (16
) recently demonstrated near-normal affinity maturation and isotype switching in LTα-KO mice after repeated challenge with high doses of antigen (NP-OVA) together with an adjuvant. As suggested by Wang et al. (17
) and illustrated by our experimental system, persistence of antigen in the context of adjuvant plausibly explains Ig affinity maturation of B cells outside germinal centers.
The magnitude of the airway inflammatory response in LTα-KO mice exposed to aerosolized OVA in the context of GM-CSF was rather striking. Indeed, whether in terms of total number of cells or of differential cell counts, the degree of the inflammatory response in the mutant mice was four- to sixfold greater than that observed in WT controls. As documented histologically (Figure ), and in agreement with Banks et al. (9
), part of the reason may be that there is an increased number of mononuclear cells in the perivascular areas of naive mutant mice. However, this difference (Figure a) appears quantitatively insufficient to explain the changes documented after exposure to our allergic sensitization protocol. A contributing factor may be that, as a result of lacking lymphoid structures (other than the spleen), there is an increased number of immune cells circulating in LTα-KO mice, as described previously by De Togni et al. (8
) and confirmed in our studies (Table ). We provide evidence of an additional, potentially important mechanism underlying the enhanced immune responsiveness observed in the mutant mice: a dramatic increase in the number of MHCII+
cells in the lung of naive mice: 29% in LTα-KO versus 7% in WT mice. That LTα-KO mice have an increased capacity for presenting antigen may also explain the very recent observation by Lee et al. (18
) of enhanced immune responses of LTα-KO mice to murine gammaherpes virus 68.
Compelled by the observation that mice lacking LN were able to generate Th2 immunity and airway eosinophilic inflammation, we investigated whether the spleen, albeit architecturally aberrant in LTα-KO mice, was able to compensate as the site of the primary immune response. The ability of splenocytes from LTα-KO mice subjected to repeated OVA aerosolizations in the context of GM-CSF to produce cytokines upon OVA recall in vitro argues for the role of the spleen in this process. That cytokine content in splenocyte cultures supernatant was, in fact, two to three times greater in LTα-KO mice than in WT control mice suggests an immune response of greater strength in the mutant mice. A decisive functional role for the spleen in allergic sensitization was provided by the splenectomy experiments. Indeed, splenectomy of LTα-KO mice fully prevented the development of airway inflammation. Our data are different from those of Davis et al. (13
), who demonstrated that removal of the spleen in LTα-KO mice decreased, but did not abrogate, humoral responses to Salmonella
in intestines. In our experiments, mice were subjected to allergic sensitization protocol 10 days after splenectomy; at this time, the peripheral cell counts of LTα-KO mice before and after splenectomy were not significantly different, indicating that the absence of sensitization and airway inflammation was not due to depletion of circulatory leukocytes and, specifically, of T cells (CD3+
) or B cells (Table ). Although these data demonstrate that the spleen indeed supplanted the role of the regional LN in LTα-KO mice, splenectomy of WT mice had no impact on the ability of asplenic mice to generate allergic sensitization and airway inflammation.
Our current understanding of the generation of mucosal immunity is that antigens penetrating mucosae are captured by DCs and transported to the draining LNs to initiate immune responses (7
). The data presented here indicate that the spleen can facilitate allergic sensitization to antigens introduced into the respiratory mucosae. This observation evokes an important question: How does aerosolized OVA reach the spleen to initiates allergic immunity? Wolvers et al. (5
) have demonstrated that after OVA delivery to the nasal mucosae, there is evidence of APCs transporting this antigen to the spleen. This DC migration may occur via the bloodstream or, presumably, through the lymphatic system, which, importantly, remains intact in LTα-KO mice. In our experimental system, OVA aerosolization leads to eosinophilic airway inflammation only in the context of GM-CSF transgene expression. Such expression is compartmentalized within the lung/airway, as GM-CSF cannot be detected in the circulation. Therefore, we surmise that it is very unlikely that exogenous GM-CSF expression in the spleen contributed to the ability of LTα-KO mice to generate allergic sensitization. Thus, our data intimate that the generation of allergic priming in the spleen of mice lacking LNs may be the result of mobilization of OVA-carrying DC from the respiratory mucosae.
In short, our data show that allergic sensitization and airway inflammation take place in the absence of thoracic (and other) LNs. That these responses are abrogated in splenectomized mice establishes the essential requirement of secondary lymphoid organs and implies that the lung does not possess the intrinsic capability to generate allergic sensitization. That the spleen can supplant the role of the draining LNs illustrates the plasticity of the immune system and highlights the systemic nature of allergic sensitization. What is the potential clinical significance of these findings? The current emphasis of the pharmacological management of asthma rests on local treatment, which effectively inhibits inflammatory processes that take place in the airway itself. However, one would argue that processes that are generated, and probably maintained, outside the airway will likely evade this treatment strategy. Thus, attempts to ultimately cure, rather than control, allergic airway inflammation may need to incorporate a systemic dimension.