Increased survival and reduced neutrophil infiltration of the liver in Rab27a- but not Munc13-4-deficient mice in lipopolysaccharide-induced systemic inflammation

Abstract

Genetic defects in the Rab27a or Munc13-4 gene lead to immunodeficiencies in humans, characterized by frequent viral and bacterial infections. However, the role of Rab27a and Munc13-4 in the regulation of systemic inflammation initiated by Gram-negative bacterium-derived pathogenic molecules is currently unknown. Using a model of lipopolysaccharide-induced systemic inflammation, we show that Rab27a-deficient (Rab27a(ash/ash)) mice are resistant to lipopolysaccharide (LPS)-induced death, while Munc13-4-deficient (Munc13-4(jinx/jinx)) mice show only moderate protection. Rab27a(ash/ash) but not Munc13-4(jinx/jinx) mice showed significantly decreased tumor necrosis factor alpha (TNF-α) plasma levels after LPS administration. Neutrophil sequestration in lungs from Rab27a(ash/ash) and Munc13-4(jinx/jinx) LPS-treated mice was similar to that observed for wild-type mice. In contrast, Rab27a- but not Munc13-4-deficient mice showed decreased neutrophil infiltration in liver and failed to undergo LPS-induced neutropenia. Decreased liver infiltration in Rab27a(ash/ash) mice was accompanied by lower CD44 but normal CD11a and CD11b expression in neutrophils. Both Rab27a- and Munc13-4-deficient mice showed decreased azurophilic granule secretion in vivo, suggesting that impaired liver infiltration and improved survival in Rab27a(ash/ash) mice is not fully explained by deficient exocytosis of this granule subset. Altogether, our data indicate that Rab27a but not Munc13-4 plays an important role in neutrophil recruitment to liver and LPS-induced death during endotoxemia, thus highlighting a previously unrecognized role for Rab27a in LPS-mediated systemic inflammation.

Fig. 1.

Kaplan-Meier survival plots for wild-type (WT), Rab27a^ash/ash (ashen), and Munc13-4 ^jinx/jinx (jinx) mice. Mice were challenged with a single intraperitoneal injection of LPS (7 mg/kg [A] or 12 mg/kg [B]). Survival curves were generated from two independent experiments with a total of 12 Rab27a^ash/ash, 12 Munc13-4^jinx/jinx, and 18 wild-type mice. The difference in survival between the wild-type and Rab27a^ash/ash mice was significant (**, P = 0.0012) by the log-rank test. Although less significant, the difference in survival between wild-type and Munc13-4^jinx/jinx mice was also evident (*, P = 0.05).

Fig. 2.

Mice lacking Rab27a but not Munc13-4 have impaired in vivo secretion of TNF-α in response to LPS. Munc13-4^jinx/jinx (jinx), Rab27a^ash/ash (ashen), and wild-type (WT) mice were challenged with a single intraperitoneal injection of LPS or PBS. Blood samples were collected at 4 h or 24 h after injection. The samples were spun down, and plasma was collected and analyzed for the presence of TNF-α as described in Materials and Methods. A total of 7 mice for each group were analyzed in three independent experiments. The statistical significance of the difference of the means was calculated using the nonparametric Mann-Whitney test.

Fig. 3.

Rab27a and Munc13-4 are not necessary for neutrophil infiltration into the lungs during LPS-induced systemic inflammation. Neutrophil infiltration into lungs was determined by analysis of tissue-associated myeloperoxidase at 4 h (A) and 24 h (B) after intraperitoneal LPS or PBS injections. Results represent the means ± SEM (n = 5 to 7 mice per group; three independent experiments). *, P < 0.01 versus PBS (nonparametric Mann-Whitney test). (C) Representative images of immunofluorescence analysis of Ly-6G-positive cells showing neutrophil infiltration into lungs of wild-type (WT), Rab27a^ash/ash (ashen), and Munc13-4^jinx/jinx (jinx) mice 4 h after LPS but not PBS administration. (D) Immunofluorescence analysis showing P-selectin expression in alveolar capillaries from Rab27a^ash/ash mice after LPS treatment. Neutrophils, visualized by staining of endogenous MPO (red), are observed both attached to the activated endothelium (arrowheads) and after transendothelial migration (arrows). (E) Immunofluorescence analysis shows expression of ICAM-1 in alveolar walls. Specific staining is also observed in the capillary endothelium (arrowheads) but not in the bronchial epithelium (arrows). Scale bars, 35 μm (C and E), 10 μm (D).

Fig. 4.

Liver sequestration of neutrophils during LPS-induced systemic inflammation requires Rab27a but not Munc13-4. Mice were injected intraperitoneally with LPS or PBS, and neutrophil infiltration into the liver was determined by analysis of tissue-associated myeloperoxidase at 4 h (A) and 24 h (B) after injections. Results are presented as means ± SEM. A total of 5 to 7 wild-type and ashen mice and 4 jinx mice for each condition were analyzed in three independent experiments. The statistical significance of the difference of the means was calculated using the nonparametric Mann-Whitney test. (C) Immunofluorescence analysis of neutrophil infiltration in liver at 4 h postinjection. Representative liver images from LPS-treated mice are shown. (D) Immunofluorescence analysis of neutrophil infiltration at 24 h postinjection of LPS. Infiltrated neutrophils (Ly-6G) are indicated with arrows. (E) Representative liver images from PBS-treated mice. Scale bars, 20 μm (C), 25 μm (E).

Fig. 5.

Effect of systemic LPS on circulating neutrophil and platelet counts in wild-type, Rab27a^ash/ash, and Munc13-4^jinx/jinx mice. Mice were treated with LPS systemically (i.p.) for 4 or 24 h. The numbers of circulating neutrophils (A) and platelets (B) were measured as described in Materials and Methods. The results are presented as means ± SEM. The statistical significance of the difference of the means was calculated using the nonparametric Mann-Whitney test (n = 3 to 6).

Fig. 6.

Effect of systemic LPS on the in vivo expression of adhesion molecules in neutrophils from wild-type, Rab27a^ash/ash, and Munc13-4^jinx/jinx mice. (A and B) Mice were injected intraperitoneally with LPS, and blood was collected 4 h after injections. The surface expression of CD11b and CD11a in wild-type (WT), Rab27a^ash/ash (ashen), and Munc13-4^jinx/jinx (jinx) neutrophils was analyzed by flow cytometry as described in Materials and Methods. Mean fluorescence intensity (MFI) is shown. (C and D) The adhesion of wild-type (WT), Rab27a^ash/ash (ashen), and Munc13-4^jinx/jinx (jinx) neutrophils to fibronectin was measured ex vivo as described in Materials and Methods. (A to D) The results are expressed as means ± SEM (n = 3 to 6 mice, three independent experiments).

Fig. 7.

Rab27a-deficient neutrophils have decreased CD44 surface expression. (A) The surface expression of the hyaluronan receptor CD44 in neutrophils was analyzed by flow cytometry. Left panel, representative histograms of CD44 expression in unstimulated neutrophils from Rab27a^ash/ash (ashen), Munc13-4^jinx/jinx (jinx), or wild-type (WT) mice after labeling with a monoclonal antibody directed at an extracellular epitope of CD44 (clone IM7) or isotype control (IC). Right panel, quantitative analysis of the MFI corresponding to CD44 expression in LPS-stimulated (100 ng/ml) and unstimulated neutrophils. Results are represented as means ± SEM (n = 6 mice). *, P < 0.03 versus the wild-type mice. (B) The adhesion of wild-type (WT) and Rab27a^ash/ash (ashen) bone marrow-derived neutrophils to hyaluronan was measured ex vivo as described in Materials and Methods. Results are means ± SEM from three independent experiments using cells from 3 wild-type and 4 ashen mice. *, P < 0.03 (nonparametric Mann-Whitney test). (C) Immunofluorescence analysis of the distribution of endogenous CD44 in ashen and wild-type neutrophils, unstimulated or treated with the chemotactic peptide fMLF. Data are representative of results of two independent experiments.

Fig. 8.

Mice lacking Rab27a or Munc13-4 have impaired in vivo secretion of MPO in response to LPS. Rab27a^ash/ash (ashen) (A) or Munc13-4^jinx/jinx (jinx) (B) and wild-type (WT) mice were challenged with a single intraperitoneal injection of LPS or PBS. Blood samples were collected at 4 h after injection. The samples were spun down, and plasma was collected and analyzed for the presence of MPO using a mouse-specific ELISA. The statistical significance of the difference of the means was calculated using the nonparametric Mann-Whitney test (n = 6 mice, three independent experiments).