In the present study, we demonstrate that Arhgef1 contributes to the development of allergic lung inflammation, and specifically establish a T-cell requirement for Arhgef1 in promoting allergic lung disease and eosinophil recruitment to the airways. These data provide further evidence for the participation of heterotrimeric G proteins and RhoA in the regulation of airway inflammation (32
), and, considering that Arhgef1 regulates the activity of both classes of G proteins, suggest that Arhgef1 contributes to this regulation. We further demonstrate that, after airway challenge within the lung, Arhgef1−/−
T cells interact in situ
with functionally normal Arhgef1−/−
antigen-presenting cells at a significantly reduced frequency, and this is reflected in the inability of antigen-specific Arhgef1−/−
T cells to produce cytokines within tissue explants after antigen stimulation. In contrast, when antigen-specific T cells and antigen-presenting cells are isolated and cultured together, efficient activation of antigen-specific mutant T cells is observed.
T cells, and specifically Th2 responses, play a predominant role in the development of AHR and associated lung inflammation (34
), and, in accord, we found an increased number of CD4+
T cells in WT lungs after sensitization and challenge. In contrast, the number of T cells found in lungs of sensitized and challenged Arhgef1−/−
mice did not differ significantly from animals that were challenged only, and is consistent with diminished inflammatory cell infiltrate and Th2 cytokines present in BAL after airway challenge of sensitized Arhgef1−/−
animals. However, Arhgef1-deficient mice harbor an increased number of all lung leukocyte cell types under both naive and inflammatory conditions, and our data indicate that antigen-specific T cells are included in this expanded population of lung leukocytes. Whether antigen-specific effector T cells are actively recruited during inflammation or are only present as a result of normal homeostatic trafficking was not addressed by these experiments. However, based on previous findings with Arhgef1-deficient marginal zone B cells and neutrophils, we speculate that mutant leukocytes accumulate in the lung as a result of impaired migration (14
Adaptive immunity is dependent on the activation of antigen-specific T cells by antigen-presenting dendritic cells and, in response to an initial antigen challenge in the airway, dendritic cells are thought to migrate to draining mediastinal lymph nodes to present captured antigen to appropriate T cells (35
). However, with subsequent antigen challenge, antigen-presenting cell–T-cell interaction within lung tissue has been shown to facilitate Th2 cytokine production and eosinophil recruitment (38
). In addition, studies in lymphotoxin-α–deficient mice, devoid of lymph nodes and Peyer's patches, demonstrate this in situ
T cell–antigen-presenting cell interaction is able to promote lung inflammation (4
). Indeed, conditional depletion of pulmonary dendritic cells suggests these cells to be both necessary and sufficient for Th2 cell stimulation during ongoing airway inflammation and after initial antigen presentation (31
). Our histologic analysis of lung tissue revealed that 48 hours after airway challenge, T cells were readily observed juxtaposed to CD11c+
antigen-presenting cells in WT lungs and similar to previous findings (38
). In contrast, the frequency with which Arhgef1−/−
T cells and antigen-presenting cells are observed to interact in mutant lungs is significantly reduced compared with WT.
The transfer of WT T cells to Arhgef1-deficient animals restored both antigen-induced AHR and inflammation, strongly suggesting that Arhgef1−/− dendritic cells are competent in capturing and presenting antigen to T cells. This was confirmed by demonstrating that antigen-pulsed bone marrow–derived Arhgef1−/− dendritic cells are able to promote AHR and leukocyte recruitment after intratracheal transfer into WT animals. Thus, in considering these data together, we suggest that the reduced interaction observed between T cells and CD11c+ cells in the lungs of mutant animals results from deficiency in T-cell function(s) in the absence of Arhgef1. However, we cannot exclude that Arhgef1−/− dendritic cell functional deficiencies exist, but are effectively compensated for by WT, but not mutant, T cells.
In response to systemic immunization with antigen, Arhgef1-deficient animals produced normal titers of antigen-specific IgE and IgG1, and generate antigen-specific splenic T cells (), demonstrating that the primary adaptive immune response leads to the appropriate activation of naive antigen-specific B and T cells. In contrast to this, previously sensitized Arhgef1 mouse mutants do not display any evidence of pulmonary inflammation in response to a subsequent airway challenge. Nevertheless, we show that Arhgef1−/− dendritic cells are functional, and that antigen-specific T cells are present in the lungs and spleens of sensitized and challenged animals. When these cell populations are isolated and cultured in vitro together with specific antigen, T cells are induced to produce cytokines and proliferate. This suggests that sensitization and challenge of mutant animals generates functional antigen-specific T cells that are present in the spleen and lungs after airway challenge. However, consistent with the lack of an in vivo immune response to airway antigenic challenge, antigen-specific Arhgef1−/− T cells in spleen and lung organ fragment cultures generated from sensitized and challenged mutants are not activated in the presence of specific antigen. From these data, we concluded that Arhgef1−/− mice mount a normal primary systemic immune response to antigenic challenge that generates antigen-specific T cells capable of migrating to the lung, but that these cells are impaired in effector function when constrained in tissue in vivo or in vitro. Impaired T-cell function in the absence of Arhgef1 is not restricted to the lung, but is also a feature of antigen-specific T cells in the spleen, suggesting that Arhgef1 is not required for a primary systemic response, but is needed by T lymphocytes for efficient activation in subsequent antigenic challenges.
What are the functional deficiencies in effector T cells in the absence of Arhgef1? Arhgef1 is a hematopoietic-restricted intracellular signaling molecule that regulates both GPCR and RhoA signaling (10
). Loss of Arhgef1 has been shown to impair neutrophil and B lymphocyte migration (14
) and, in particular for marginal zone B cells, leads to aberrant resolution of integrin adhesion during migration (15
). We have not yet satisfactorily established similar deficiencies in Arhgef1−/−
T cells, but the concept of Arhgef1 playing a similar role in T cells, or a T-cell subset, is supported by the reduced frequency in which T cells and CD11c+
cells are found to interact in the lung and the inability of these cells to be activated in vivo
or in vitro
organ cultures where migration of these antigen-specific T cells on integrin ligands would be necessary. In contrast, in vitro
cultures of isolated lung leukocytes (i.e, cell suspensions) from sensitized and challenged Arhgef1−/−
animals demonstrate that mutant T cells can be stimulated to produce Th2 cytokines in an antigen-specific manner. These data demonstrate that antigen-specific Arhgef1−/−
T cells are functional when isolated and cultured together with resident antigen-presenting cells, but not when these same cells are constrained by tissue, either in vivo
or in vitro
. Although speculative, we interpret these findings to suggest that aberrant effector T-cell migration within lung tissue underscores the reduced frequency with which Arhgef1−/−
T cells interact with antigen-presenting cells and, consequently, precludes efficient antigen-specific T-cell activation. Furthermore, although we have previously suggested Arhgef1 activation of RhoA to be important in the regulation of integrin adhesion during migration, given the ability of Arhgef1 to also regulate GPCR signaling, Arhgef1−/−
T cells may also be impaired in responding to dendritic cell–derived chemoattractants.
In summary, these data document an important role for Arhgef1 in the development of airway hyperreactivity and inflammation, and further demonstrate that effector T cells are dependent on Arhgef1 for promoting an inflammatory response. Moreover, we provide additional evidence that suggests that the inability of antigen-specific T cells to facilitate allergic lung disease is a consequence of inefficient interaction between the mutant T cells and functional CD11c+ cells in the Arhgef1−/− lung. Thus, Arhgef1, a regulator of Gα12/13 and RhoA signaling, plays an important role in regulating effector T-cell function required for the development of airway inflammation.