Dose-dependent activation of lymphocytes in endotoxin-induced airway inflammation

Abstract

Recruitment of neutrophils to lung tissue and airspaces is a hallmark of inflammatory events following inhalation of endotoxins. We studied the role of different lymphocyte subsets in this inflammation, which is assumed to primarily involve the innate immune system. Inhalation of aerosolized Escherichia coli lipopolysaccharide (LPS) in mice induced a dose-dependent increase in neutrophils in bronchoalveolar lavage fluid, reaching a maximum after 12 h at a low dose and after 24 h at a high dose. Profiles of gene expression in lung tissue indicated an early (2 h) and transient onset of proinflammatory cytokines and chemokines by a low dose of LPS, while a high dose caused more delayed and sustained (6 to 12 h) activation. Gamma interferon, interleukin-2 (IL-2), RANTES, and the alpha chain of the IL-2 receptor were not expressed at a low dose, whereas a high dose of LPS induced a strong expression of these genes, indicating a dose-dependent activation of T cells. A similar pattern was observed for IL-17, supporting a contribution of T cells to the neutrophilic inflammation only at high-dose exposure to LPS. The involvement of lymphocytes in the inflammatory response was further studied using mice with functional deficiencies in defined lymphocyte subsets. Both gammadelta T-cell- and B-cell-deficient mice displayed a response similar to that of the corresponding wild-type strains. Selective depletion of NK cells by in vivo administration of the pk136 antibody did not significantly affect the recruitment of neutrophils into airspaces. Thus, neither NK cells, B cells, nor gammadelta T cells appeared to participate in the host response, suggesting that among the lymphocyte subsets, alphabeta T cells are exclusively involved in endotoxin-induced airway inflammation.

FIG. 1

Dose-dependent induction of airway inflammation by aerosolized LPS. Withdrawal and analysis of BALF was performed 16 h after LPS exposure. The total number of leukocytes was determined by cell counting, and the proportion of granulocytes was analyzed by flow cytometric straining using the GR-1 MAb. Control mice (0 μg of LPS per ml) were exposed to an aerosol of solvent alone (endotoxin-free distilled water). The leukocytes recovered from these mice were predominantly alveolar macrophages (>95%), with a proportion of granulocytes generally less than 5%. At nebulizer concentrations of 100 and 1,000 μg/ml, the numbers of granulocytes and total leukocytes were significantly increased in BALF compared to that of animals not exposed to LPS. ∗∗, P < 0.01; ∗∗∗, P < 0.001. Mean values and SEM are shown (three to five animals in each group).

FIG. 2

Kinetics for the inflammatory response after inhalation of low-dose (100 μg/ml) and high-dose (1,000 μg/ml) LPS. The total number of leukocytes in BALF was determined by cell counting, and the number of granulocytes was determined by flow cytometry staining using the GR-1 MAb. Mean values and SEM are shown (four animals in each group).

FIG. 3

Expression profiles for chemokines (a and b), proinflammatory cytokines (c and d), and immunoregulatory cytokines and IL-2Rα (e and f) after exposure to low-dose (a, c, and e) and high-dose (b, d, and f) LPS. Expression of mRNA was semiquantitatively analyzed in lung tissue at different time points after LPS exposure. The relative amount of each transcript is indicated in relation to the corresponding amount of the housekeeping gene that encodes GAPDH.

FIG. 4

The inflammatory response in KO mice lacking γδ T cells. The total number of granulocytes in BALF was analyzed 16 h after LPS exposure. The response did not differ significantly between the TCR-δ^−/− and the corresponding wild-type strain, at either low- or high-dose exposure. Mean values and SEM are shown (three to five animals in each group).

FIG. 5

The inflammatory response in B-cell-deficient mice. The total number of granulocytes in BALF was analyzed 16 h after LPS exposure in B-cell KO mice (the Igh-6^tm1Cgn strain) and in the corresponding wild-type strain (C57BL/10). The response did not differ significantly between these two strains, at either low- or high-dose exposure. Unexposed animals from the B-cell KO strain did not display detectable airway inflammation (not shown). Mean values and SEM are shown (three to five animals in each group).

FIG. 6

Depletion of NK cells in lungs by treatment with the pk136 MAb. Mice were injected i.p. either with 200 μg of pk136 or with solvent alone (PBS) 2 days before LPS exposure. Withdrawal of cells in airspaces was performed 16 h after inhalation of LPS. Flow cytometric staining of NK cells in BALF from untreated wild-type C57BL/6 (a) and TCR-αβ/γδ double KO mice (c) is illustrated. Data for mice treated with the pk136 MAb are shown in panels b (wild type) and d (TCR-αβ/γδ double KO). Gates were set for lymphocytes in forward scatter and side scatter, and within the lymphocyte population CD3-negative cells expressing NK1.1 were identified as NK cells (upper left quadrant). In BALF of T-cell-deficient mice treated with pk136, only scarce lymphocytes were found, in contrast to the large number of lymphocytes, predominantly NK cells, found in untreated mice. The proportion of NK cells in BALF was smaller in wild-type C57BL/6 mice than in T-cell KO mice (<1% of total BALF leukocytes, compared to about 5% in TCR-β^−/−δ^−/− mice). FITC, fluorescein isothiocyanate. PE, phycoerythrin.

FIG. 7

Unaffected airway inflammation in mice depleted of NK cells. C57BL/6 mice were injected i.p. with 200 μg of anti-NK1.1 MAb (pk136) and 2 days thereafter were exposed to aerosolized LPS at a high or low dose. Withdrawal of BALF cells was performed 16 h after inhalation of LPS. The number of granulocytes in BALF did not differ significantly between the groups of NK-cell-depleted mice and the corresponding groups of control mice injected with solvent alone (PBS). Mean values and SEM are shown (four to five animals in each group).