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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2010 Feb;176(2):926–938. doi: 10.2353/ajpath.2010.090342

Mast Cell-Derived TNF Can Exacerbate Mortality during Severe Bacterial Infections in C57BL/6-KitW-sh/W-sh Mice

Adrian M Piliponsky *, Ching-Cheng Chen *, Michele A Grimbaldeston *†, Stacy M Burns-Guydish ‡, Jonathan Hardy §, Janet Kalesnikoff *, Christopher H Contag §¶, Mindy Tsai *, Stephen J Galli *
PMCID: PMC2808097  PMID: 20035049

Abstract

We used mast cell-engrafted genetically mast cell-deficient C57BL/6-KitW-sh/W-sh mice to investigate the roles of mast cells and mast cell-derived tumor necrosis factor in two models of severe bacterial infection. In these mice, we confirmed findings derived from studies of mast cell-deficient WBB6F1-KitW/W-v mice indicating that mast cells can promote survival in cecal ligation and puncture (CLP) of moderate severity. However, we found that the beneficial role of mast cells in this setting can occur independently of mast cell-derived tumor necrosis factor. By contrast, using mast cell-engrafted C57BL/6-KitW-sh/W-sh mice, we found that mast cell-derived tumor necrosis factor can increase mortality during severe CLP and can also enhance bacterial growth and hasten death after intraperitoneal inoculation of Salmonella typhimurium. In WBB6F1-KitW-sh/W-sh mice, mast cells enhanced survival during moderately severe CLP but did not significantly change the survival observed in severe CLP. Our findings in three types of genetically mast cell-deficient mice thus support the hypothesis that, depending on the circumstances (including mouse strain background, the nature of the mutation resulting in a mast cell deficiency, and type and severity of infection), mast cells can have either no detectable effect or opposite effects on survival during bacterial infections, eg, promoting survival during moderately severe CLP associated with low mortality but, in C57BL/6-KitW-sh/W-sh mice, increasing mortality during severe CLP or infection with S. typhimurium.

The factors determining whether particular infections will be successfully controlled or progress to death are incompletely understood. Studies conducted using genetically mast cell-deficient (WB/ReJ × C57BL/6)F1-KitW/W-v mice (WBB6F1-KitW/W-v mice), the corresponding normal (WBB6F1-Kit+/+) mice, and mast cell-engrafted WBB6F1-KitW/W-v mice have indicated that mast cells can increase survival during various models of bacterial infection of moderate severity,1,2,3,4,5,6,7,8,9,10,11,12,13 defined herein as infections that result in relatively low mortality in normal mice. Although mast cells can contribute to host defense against bacteria by multiple direct and indirect mechanisms,1,2,3,4,5,7,8,9,10,11,12,13 several groups have focused on the potential role of mast cell-derived tumor necrosis factor (TNF) in such settings.1,2,3,4,5,7 Results obtained in work using TNF-deficient mice4 or mice in which the actions of TNF are blocked by neutralizing antibodies1,2 have clearly demonstrated that TNF can have protective functions during some bacterial infections, and that such TNF-dependent effects may include the enhancement of neutrophil recruitment and/or function and the promotion of bacterial clearance.

However, such genetic or neutralizing antibody-based approaches eliminate or reduce the function of TNF derived from all cellular sources, not just mast cell-derived TNF. Thus, there has been no direct evidence yet published that shows that mast cells represent a critical source of TNF in such settings. Moreover, WBB6F1-KitW/W-v mice have a modest deficiency in neutrophils,14,15,16 and this abnormality, as well as their virtual lack of mast cells, might contribute to the increased pathology observed in these mice during certain models of bacterial infection. Therefore, in the present study, we used genetically mast cell-deficient C57BL/6-KitW-sh/W-sh mice (produced as in ref.17) engrafted with either wild-type mast cells or mast cells unable to produce TNF to re-examine the roles of mast cells, and mast cell-derived TNF, in two different models of bacterial infection.

Adult C57BL/6-KitW-sh/W-sh mice are profoundly mast cell-deficient,17,18,19 as are WBB6F1-KitW/W-v mice,20,21 but, unlike WBB6F1-KitW/W-v mice, C57BL/6-KitW-sh/W-sh mice have been reported to have increased levels of blood and bone marrow neutrophils.15,16 We used both WBB6F1-KitW/W-v mice and C57BL/6-KitW-sh/W-sh mice to investigate the role of mast cells in moderately severe versus severe cecal ligation and puncture (CLP), with moderately severe versus severe CLP defined herein as models of CLP in which no more than 50% (in the moderately severe CLP model) vs. more than 50% (in the severe CLP model) of normal mice succumb during the first 4 days after surgery to induce acute bacterial peritonitis. Our observations in C57BL/6-KitW-sh/W-sh mice confirm work in WBB6F1-KitW/W-v mice1,3,4,5,6,8 in providing evidence that mast cells can enhance host resistance and survival during moderately severe CLP, a well known model of mouse bacterial peritonitis, but provide evidence that mast cells can enhance survival in this model independently of their ability to produce TNF. By contrast, mast cell-derived TNF increased mortality during severe CLP in C57BL/6-KitW-sh/W-sh mice. We found that mast cell-derived TNF also can hasten mortality in C57BL/6-KitW-sh/W-sh mice subjected to a different model of severe bacterial infection: that induced by the intraperitoneal inoculation of Salmonella typhimurium.

Materials and Methods

Mice

c-kit mutant genetically mast cell-deficient (WB/ReJ-KitW/+ × C57BL/6J-KitW-v/+) F1-KitW/W-v (WBB6F1-KitW/W-v or KitW/W-v) mice and the congenic normal WBB6F1+/+ (WBB6F1- Kit+/+) mice, and C57BL/6J (B6J) mice, were purchased from Jackson Laboratories (Bar Harbor, ME). The Wsh or “sash” mutation was named after the broad white belt or sash observed on the coat of a single female offspring of a cross between two inbred strains (C3H/HeH × 101/H).22 Wsh homozygotes were characterized as black-eyed white mice (some of them with patches of pigment on the ear pinnae and/or base of the tail) that were viable, non-anemic and fertile,22 but which had a marked reduction in mast cells in multiple anatomical sites.23 Genetically mast cell-deficient C57BL/6-KitW-sh/+ mice (B6-KitW-sh/+ mice)24 were the generous gift of Peter Besmer (Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY); while these mice were reported to be on the C57BL/6 background, it is not clear from the publication whether, and if so how many times, these mice had been backcrossed to mice from the Jackson Laboratories’ C57BL/6J population. We used such B6-KitW-sh/+ mice to produce B6-KitW-sh/W-sh mice for some of our initial CLP experiments; for these experiments, we used the B6-Kit+/+ littermates as wild-type controls. In addition, we backcrossed B6-KitW-sh/+ mice with C57BL/6J (B6J) mice for 11 generations to produce B6J-KitW-sh/W-sh mice; for CLP experiments using these mice, we used B6J-Kit+/+ mice purchased from Jackson Laboratories as wild-type controls. Like B6-KitW-sh/W-sh mice,17,19 we found that adult (∼12-week-old) B6J- KitW-sh/W-sh mice lacked any detectable mast cells in the peritoneal cavity or mesentery. Based on reverse transcription-PCR analysis and sequencing of the resultant PCR products, we confirmed that the Wsh inversion 3′ breakpoint in our B6J-KitW-sh/W-sh mice and that in the B6-KitW-sh/W-sh mice that were characterized by Nigrovic et al16 are identical (data not shown). To generate WB/ReJ-KitW-sh/+ mice, B6J-KitW-sh/+ mice were crossed with WB/ReJ-Kit+/+ mice for 11 generations. Some genetically mast cell-deficient B6J-KitW-sh/+ mice were then crossed with WB/ReJ-KitW-sh/+ mice to produce WBB6F1- KitW-sh/W-sh mice (WB/ReJ-KitW-sh/+ × B6-KitW-sh/+) F1-KitW-sh/W-sh and the normal wild-type littermates (ie, WBB6F1-Kit+/+ mice). TNF-deficient mice (Tnf−/−) on the C57BL/6J background25 were bred and maintained at the Stanford University Research Animal Facility. Unless specified otherwise, all experiments were performed using 12-week-old male mice. All animal care and experimentation was conducted in accord with current National Institutes of Health guidelines and with the approval of the Stanford University Institutional Animal Care and Use Committee.

Mast Cell Engraftment of Mast Cell-Deficient Mice

Some WBB6F1-KitW/W-v, B6-KitW-sh/W-sh, and WBB6F1- KitW-sh/W-sh mice (4 to 6 weeks old) were repaired of their mast cell deficiency selectively and locally by the i.p. injection of growth factor-dependent, congenic mouse bone marrow-derived cultured mast cells (BMCMCs). Briefly, femoral bone marrow cells from WBB6F1-Kit+/+ or B6-Kit+/+ mice were maintained in vitro for ∼4 weeks in interleukin-3 (Peprotech, Rocky Hill, NJ)-containing medium until mast cells represented >95% of the total cells according to staining with May-Grünwald-Giemsa. 2.0 × 106 BMCMCs, in 200 μl of PBS, were injected i.p. (via a 26G needle) and the mice were used for experiments, together with strain-, gender-, and age-matched mast cell-deficient mice, 4 to 6 weeks after adoptive transfer of BMCMCs. Other B6-KitW-sh/W-sh mice received, 4 to 6 weeks before CLP, 2.0 × 106 BMCMCs generated from B6-Tnf−/− mice. The numbers of peritoneal mast cells were similar in C57BL/6-KitW-sh/W-sh mice that had been engrafted with either wild-type or TNF-deficient mast cells (3.8 + 0.8% or 3.6 + 0.7% of total cells) or in WBB6F1-KitW-sh/W-sh mice that had been engrafted with WBB6F1 wild-type mast cells (6.1 + 0.5% of total cells), as was the distribution of mast cells in the mesentery (ie, mesenteric windows) of these three groups of mice (6.2 + 0.96, 6.4 + 0.64, or 6.1 + 0.55 mast cells/mm2, respectively).

Infection with S. typhimurium

The wild-type virulent S. typhimurium strain, SL1344, was obtained from Bruce Stocker (deceased) of Stanford University and labeled with the Lux operon to create bioluminescent SL1344lux (SMB500) as reported.26 Bacteria were grown as previously described27 and mice were infected by i.p. injection of 25 colony forming units (CFUs) of the labeled (ie, bioluminescent) bacteria.

In Vivo Bioluminescence Imaging

Mice were imaged as previously described.27 Briefly, mice were placed in an in vivo Imaging System (IVIS, a Xenogen product from Caliper LifeSciences, Alameda, CA). A grayscale reference image was taken under low light illumination followed by a 5 minutes image, in complete dark, of the bioluminescent signal originating from the labeled bacteria within the animals. The data were expressed as photons emitted per mouse, or region of interest, per second. A pseudocolor representation of the photon collection (red signifying most intense and blue signifying least intense) was superimposed over the grayscale reference image. Acquisition and analysis of data were performed using the LivingImage (a Xenogen product from Caliper LifeSciences) software, which runs as an overlay on the IgorPro image analysis package (Wavemetrics, Lake Oswego, OR). Mice were imaged before and at selected time points after infection.

CLP

CLP was performed as described.1 Briefly, mice were deeply anesthetized by i.m. injection of 100 mg/kg ketamine and 20 mg/kg xylazine. The cecum was exposed by a 1- to 2-cm midline incision on the anterior abdomen and subjected to ligation of its distal portion and a single needle puncture of the ligated segment. Protocols for eliciting severe or moderately severe CLP were developed so that >50% or 20% to 50% of the wild-type mice died within 4 days after CLP. We used survival as the criterion to classify the severity of CLP, identifying procedures of performing “moderately severe” or “severe” CLP, which resulted in the death of no more than 50% or >50% of wild-type mice, respectively. However, we found, as expected, that the hypothermia induced in wild-type mice by CLP was more pronounced in mice subjected to severe CLP than in mice subjected to moderately severe CLP (a reduction in body temperature of 4.6 + 0.86°C vs. 1.6 + 0.48°C [mean + SD] for severe vs. moderately severe CLP, P < 0.05, n = 5/group) at 18 to 24 hours after surgery.

For severe CLP, we ligated the distal half or two thirds of the cecum and made a single puncture with a 22G needle; for moderately severe CLP, we ligated the distal half of the cecum and made a single puncture with a 22G needle. The cecum was then replaced into the abdomen, 1 ml of sterile saline (pyrogen-free 0.9% NaCl) was administrated into the peritoneal cavity, and the incision was closed using 9-mm steel wound clips. Mice were observed for mortality at least four times daily. Mice that were clearly moribund were euthanized by CO2 inhalation.

Quantification of Leukocytes and Platelets

Absolute numbers of neutrophils and platelets in the blood were counted using the Abbott Cell-Dyn 3500 automated hematology analyzer. Neutrophils (Gr-1high/F4/80− cells) in the bone marrow, spleen and peritoneal fluid, macrophages (Gr-1−/F4/80+ cells) in the peritoneal fluid, and basophils (FcεRI+/c-Kit−/DX5+ cells) in the blood were analyzed by flow cytometry. Briefly, red cells were lysed with AKC lysis buffer for 5 minutes. Total cell numbers were counted using a hemocytometer. Cells were blocked with unconjugated anti-CD16/CD32 on ice for 5 minutes and then stained with a combination of fluorescein isothiocyanate-labeled anti-Gr-1 (RB6-8C5, 2.5 μg/ml) and allophycocyanin-labeled anti-F4/80 (BM8, 4 μg/ml of each) antibodies (eBioscience San Diego, CA) on ice for 15 minutes for the detection of neutrophils and macrophages. To identify basophils (as c-Kit−CD49b+FcεRI α chain+ cells28), we used a combination of phycoerythrin-labeled anti-c-Kit (2B8, 1 μg/ml) (Pharmingen, San Jose CA), allophycocyanin-labeled anti-CD49b (DX5, 8 μg/ml), and fluorescein isothiocyanate-labeled anti-FcεRI α chain (MAR-1, 2.5 μg/ml) (e-Bioscience) antibodies. The expression of cell surface markers was analyzed on a FACSCaliber (BD Biosciences) and using FlowJo Software (version 5.7.2). Gates for subpopulations of cells were based on unstained cells, as well as single color stain of the cells, to determine compensation and nonspecific fluorescence. Propidium iodide was used to detect dead cells. Only cells negative for propidium iodide were used for analysis.

Quantification of Bacterial CFUs

Dilutions of peritoneal fluids were performed and samples plated on Luria Bertani (LB) agar. Colonies were counted after overnight incubation at 37°C.

Measurement of TNF

TNF levels in the supernatants were assessed by enzyme-linked immunosorbent assay (BD OptEIA ELISA [BD Biosciences]. The detection limit for this assay is 15 pg/ml.

Reverse Transcription-PCR Analysis

The KitW-sh inversion mutation in our B6J-KitW-sh/W-sh mice was assessed by PCR using PCR primers with the sequences described by Nigrovic et al16 (which flank the reported KitW-sh inversion 3′ breakpoint).

Statistical Analysis

We assessed differences in the survival rates after CLP using the Mantel-Haenszel log-rank test. All other data were analyzed for statistical significance using the Mann Whitney U-test. P < 0.05 is considered statistically significant. Unless otherwise specified, all data are presented as mean ± SEM.

Results

Increased Mortality of Mast Cell-Deficient WBB6F1-KitW/W-v Mice after Severe CLP Is Not Reduced by Mast Cell Engraftment

When we subjected WBB6F1 mice to moderately severe CLP, we confirmed findings first reported by Echtenacher et al1: the survival of genetically mast cell-deficient WBB6F1-KitW/W-v mice (15% at day 7) was significantly worse than that of the corresponding wild-type mice (50% survival at day 7; P = 0.0072) or WBB6F1-KitW/W-v mice that had been selectively engrafted with wild-type bone marrow-derived cultured mast cells (ie, Kit+/+ BMCMCs→KitW/W-v mice) via i.p. transfer (60% survival at day 7, P = 0.017) (Figure 1A). Mast cell-deficient WBB6F1-KitW/W-v mice also exhibited higher mortality than the corresponding wild-type mice in a severe model of CLP (0 vs. 29% survival at days 3 or 7 after CLP, respectively, P = 0.032) (Figure 1B). However, the survival of Kit+/+ BMCMCs→KitW/Wv mice subjected to severe CLP was similar to that observed in mast cell-deficient KitW/W-v mice (P = 0.62), suggesting that the reduced survival of WBB6F1-KitW/W-v mice in this model may have reflected c-kit-related abnormalities other than, or in addition to, their mast cell deficiency.

Figure 1.

Figure 1

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Mast cells enhance survival in WBB6F1-KitW/W-v mice after moderately severe but not severe CLP. A: Survival after moderately severe CLP (50% ligation; single puncture with 22G needle) in wild-type (WBB6F1-Kit+/+) mice (black squares) (n = 8), WBB6F1-KitW/W-v mast cell-deficient mice (white squares) (n = 13) and WBB6F1-Kit+/+ BMCMCs→WBB6F1-KitW/W-v mice (black circles) (n = 10). B: Survival after severe CLP (2/3 ligation; single puncture with 22G needle) in wild-type (WBB6F1-Kit+/+) mice (black squares) (n = 7), WBB6F1-KitW/W-v mast cell-deficient mice (white squares) (n = 10) and WBB6F1-Kit+/+ BMCMCs→WBB6F1-KitW/W-v mice (black circles) (n = 10). *P < 0.05, **P < 0.01 versus KitW/W-v mice; §P = 0.076, †P < 0.05, †††P < 0.001 versus corresponding results for moderately severe CLP. All mice were females that were 12 weeks old at the beginning of the experiment. n.s., not significant.

These results are concordant with those of our previous study of a different model of a severe bacterial infection, that induced by the intraperitoneal injection of S. typhimurium.27 In that study, we found that mast cell-deficient WBB6F1-KitW/W-v mice exhibited significantly more bacterial CFUs per spleen at day 6 of S. typhimurium infection, as well as decreased survival, as compared with the corresponding wild-type mice.27 However, the abnormalities in the response of WBB6F1-KitW/W-v mice were not corrected in Kit+/+ BMCMCs→KitW/W-v mice, indicating that c-kit related factors in WBB6F1- KitW/W-v mice other than the lack of mast cells, perhaps including defects in their neutrophil responses,14,15,16 may have contributed to their reduced survival in this model.

Mast Cells Can Enhance Survival after Moderately Severe CLP Independently of Their Ability to Produce TNF

We also investigated the role of mast cells in moderately severe versus severe CLP using C57BL/6-KitW-sh/W-sh mice. Some experiments assessing moderately severe or severe CLP were performed with B6-KitW-sh/W-sh mice and others using B6J-KitW-sh/W-sh mice; the results obtained with each of these groups were very similar and have been combined for presentation in the Figures. In contrast to mast cell-deficient WBB6F1-KitW/W-v mice, mast cell-deficient B6-KitW-sh/W-sh or B6J-KitW-sh/W-sh mice (which we will refer to here as C57BL/6-KitW-sh/W-sh mice) have increased numbers of neutrophils (identified in flow cytometry as Gr1hi, F4/80− cells) in the blood, bone marrow, and spleen (15,16 and Supplemental Figure S1 at http://ajp.amjpathol.org). Moreover, since these mice have the C57BL/6 background, they can be engrafted with wild-type or TNF-deficient mast cells of the C57BL/6 strain background to assess the extent to which TNF derived from mast cells as opposed to other cell types contributes to the outcome of moderately severe and severe CLP. This may be an important consideration, as evidence has been reported indicating that mast cells from mice of different strain backgrounds can differ in their expression of cell surface structures29 and in their responses to activation via FcεRI.30

Like WBB6F1-KitW/W-v mice, mast cell-deficient C57BL/6-KitW-sh/W-sh mice exhibited reduced survival after moderately severe CLP when compared with the congenic normal mice (16 vs. 61% survival at day 7, respectively, P = 0.028) (Figure 2A). However, there were no statistically significant differences in survival after moderately severe CLP for wild-type mice or C57BL/6-KitW-sh/W-sh mice that had been engrafted with either wild-type (Tnf+/+) mast cells (Tnf+/+ BMCMCs→KitW-sh/W-sh mice) or TNF-deficient (Tnf−/−) mast cells (Tnf−/− BMCMCs→KitW-sh/W-sh mice) (Figure 2A). These results therefore do not support the hypothesis that mast cells have to be able to produce TNF to enhance survival after moderately severe CLP.

Figure 2.

Figure 2

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Mast cells, but not mast cell-derived TNF, contribute to intraperitoneal neutrophil numbers, bacterial clearance, and survival after moderately severe CLP in C57BL/6-KitW-sh/W-sh mice. A: Survival after moderately severe CLP (50% ligation; single puncture with 22G needle) in wild-type C57BL/6 (B6-Kit+/+) mice (black squares) (n = 14), B6-KitW-sh/W-sh mast cell-deficient mice (white squares) (n = 17), B6-Tnf+/+ BMCMCs→KitW-sh/W-sh mice (blue circles) (n = 18) and B6-Tnf−/− BMCMCs→B6-KitW-sh/W-sh mice (yellow rhomboids) (n = 15). B–E: Numbers of neutrophils (polymorphonuclear leukocytes [PMN]) (B) amount of TNF (C), and numbers of bacterial CFUs (D), in the peritoneal lavage fluid at 18 to 24 hours after moderately severe CLP in wild-type C57BL/6 mice (B6-Kit+/+) (n = 5 to 7), B6-KitW-sh/W-sh mast cell-deficient mice (n = 5 to 16), B6-Tnf+/+ BMCMCs→B6-KitW-sh/W-sh mice (n = 8 to 13), and B6-Tnf−/− BMCMCs→B6-KitW-sh/W-sh mice (n = 5 to 7). In (A), *P < 0.05 versus B6 mice; in (B–D), *P < 0.05, **P < 0.01 for the comparisons indicated. All mice were males that were 12 weeks old at the beginning of the experiment. n.s., not significant.

Several lines of evidence indicate that one mechanism by which mast cells can augment innate immunity during bacterial infections is by initiating and augmenting leukocyte recruitment, thereby enhancing pathogen clearance.1,2,12,31,32 At 18 to 24 hours after moderately severe CLP, C57BL/6-KitW-sh/W-sh mice, which had been engrafted with either wild-type or TNF-deficient mast cells, contained increased numbers of peritoneal neutrophils (Figure 2B) as compared with mast cell-deficient C57BL/6-KitW-sh/W-sh mice. Although all groups of mice exhibited very similar amounts of TNF in the peritoneal cavity at 18 to 24 hours after CLP (Figure 2C), only mast cell-deficient C57BL/6-KitW-sh/W-sh mice (the group with the lowest levels of intraperitoneal neutrophils [Figure 2C]) exhibited high numbers of intraperitoneal bacterial CFUs at that time (Figure 2D). These results suggest that mast cells can have effects that increase numbers of intraperitoneal neutrophils, and bacterial clearance, in this model independently of the mast cell’s ability to influence levels of TNF.

It has been reported that in some models of intraperitoneal inflammation,2,33 mast cells represent an early source of TNF. We therefore also measured TNF levels in the peritoneum of wild-type C57BL/6 mice versus mast cell-deficient C57BL/6-KitW-sh/W-sh mice at 6 hours after moderately severe CLP. We found that while the levels of intraperitoneal TNF were substantially lower in both groups than when measured at 18 to 24 hours after CLP (compare results in Figure 2C and Supplemental Figure S1A at http://ajp.amjpathol.org), the levels in the mast cell-deficient C57BL/6-KitW-sh/W-sh mice were only slightly (but not significantly) lower than those in the wild-type mice. Similarly, we did not observe significant differences in the numbers of neutrophils in the peritoneal cavity of wild-type (C57BL/6-Kit+/+) vs. C57BL/6-KitW-sh/W-sh mast cell deficient mice at 6 hours after moderately severe CLP (Supplemental Figure S1B at http://ajp.amjpathol.org). While these data do not rule out the possibility that mast cells may represent one source of “early TNF” in the moderately severe CLP model in C57BL/6 mice, they do indicate that any such effects are relatively modest in this setting.

Mast Cell-Derived TNF Can Impair Survival after Severe CLP in C57BL/6-KitW-sh/W-sh Mice

In contrast to our findings in WBB6F1-KitW/W-v mice (Figure 1B), mast cell-deficient C57BL/6-KitW-sh/W-sh mice exhibited significantly better survival than the corresponding wild-type mice after severe CLP (P = 0.029) (Figure 3A). The survival of Tnf+/+ BMCMCs→KitW-sh/W-sh mice was not significantly different from that of C57BL/6 wild-type mice (P = 0.25) (Figure 3A), and the survival of Tnf−/− BMCMCs→KitW-sh/W-sh mice was not significantly different from that of mast cell-deficient C57BL/6- KitW-sh/W-sh mice (P = 0.45). Overall, these data show that wild-type mast cells, but not TNF-deficient mast cells, can significantly impair the survival of C57BL/6-KitW-sh/W-sh mice subjected to severe CLP.

Figure 3.

Figure 3

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Mast cells, and mast cell-derived TNF, increase intraperitoneal neutrophil numbers and amounts of intraperitoneal TNF, but reduce survival, after severe CLP in C57BL/6-KitW-sh/W-sh mice. A: Survival after severe CLP (2/3 ligation; single puncture with 22G needle) in wild-type C57BL/6 mice (B6-Kit+/+) (black squares) (n = 19), B6-KitW-sh/W-sh mast cell-deficient mice (white squares) (n = 23), B6-Tnf+/+ BMCMCs→B6-KitW-sh/W-sh mice (blue circles) (n = 20) and B6-Tnf−/− BMCMCs→B6-KitW-sh/W-sh mice (yellow rhomboids) (n = 20). B–D: Numbers of neutrophils (polymorphonuclear leukocytes [PMN]) (B), amount of TNF (C), and numbers of bacterial CFUs (D), in the peritoneal lavage fluid at 18 to 24 hours after severe CLP in wild-type C57BL/6 mice (B6-Kit+/+) (n = 7 to 11), B6-KitW-sh/W-sh mast cell-deficient mice (n = 7 to 16), B6-Tnf+/+ BMCMCs→B6-KitW-sh/W-sh mice (n = 9 to 13), and B6-Tnf−/− BMCMCs→B6-KitW-sh/W-sh mice (n = 5 to 8). In (A), *P < 0.05 versus B6-KitW-sh/W-sh mice; §§P < 0.01 vs. B6-Kit+/+ mice or B6-Tnf+/+ BMCMCs→B6-KitW-sh/W-sh mice; in (B–D). *P < 0.05, **P < 0.01, ***P < 0.001 for the comparisons indicated; †P < 0.05, ††P < 0.01, †††P < 0.001, ††††P < 0.0001 versus corresponding results for moderately severe CLP. Survival curves for B6-KitW-sh/W-sh mast cell-deficient mice and B6-Tnf−/− BMCMCs→B6-KitW-sh/W-sh mice were P = 0.17 and P = 0.11 versus corresponding results for moderately severe CLP. All mice were males that were 12 weeks old at the beginning of the experiment. n.s., not significant.

When assessed at 18 to 24 hours after severe CLP, C57BL/6-KitW-sh/W-sh mice engrafted with wild-type mast cells exhibited significantly higher numbers of neutrophils (Figure 3B) and amounts of TNF (Figure 3C) in the peritoneal cavity than did C57BL/6-KitW-sh/W-sh mice engrafted with Tnf−/− mast cells; numbers of bacterial CFUs were also higher, although this result was not statistically significant (P = 0.21) (Figure 3D). The two groups with the worst survival after severe CLP (ie, wild-type mice and C57BL/6-KitW-sh/W-sh mice engrafted with wild-type mast cells) also had significantly higher numbers of neutrophils, amounts of TNF and numbers of bacterial CFUs in the peritoneal cavity than did the corresponding groups subjected to moderately severe CLP (compare Figure 2, B–D with Figure 3, B–D). By contrast, the two groups with the best survival after severe CLP (ie, mast cell-deficient C57BL/6-KitW-sh/W-sh mice and C57BL/6-KitW-sh/W-sh mice engrafted with Tnf−/− mast cells) had amounts of TNF and numbers of bacterial CFUs in the peritoneal cavity 18 to 24 hours after severe CLP that did not differ significantly from the corresponding values in the same groups of mice during moderately severe CLP (compare Figure 2, C and D, with Figure 3, C and D).

Our findings demonstrate that, in C57BL/6-KitW-sh/W-sh mice, mast cells have effects that can significantly increase levels of TNF during severe CLP (whether by producing the TNF themselves or by influencing the ability of other cells to do so), but not during moderately severe CLP (compare Figure 2C with Figure 3C). Our data also show that such effects are associated with (and possibly may contribute to) higher levels of neutrophil recruitment/retention in the severe CLP model, even though mast cell-derived TNF does not appear to contribute to the mast cell-dependent increase in intraperitoneal neutrophils during moderately severe CLP (compare Figure 2B with Figure 3B).

In WBB6F1-KitW-sh/W-shMice, Mast Cells Can Significantly Enhance Survival in Moderately Severe CLP but Not in Severe CLP

To assess the extent to which mouse strain background might influence differences in responses to CLP in WBB6F1-KitW/W-v versus C57BL/6-KitW-sh/W-sh mice, we generated WBB6F1-KitW-sh/W-sh mice. We first compared numbers of various leukocyte populations and blood platelets, and spleen size, in WBB6F1-KitW/W-v mice, B6J-KitW-sh/W-sh mice, and WBB6F1-KitW-sh/W-sh mice and the corresponding wild-type Kit+/+ mice (Supplemental Figures 2–6 at http://ajp.amjpathol.org).

Like naïve B6-KitW-sh/W-sh mice16 and B6J-KitW-sh/W-sh mice, WBB6F1-KitW-sh/W-sh mice exhibited increased numbers of neutrophils (Gr1+/hi, F4/80− cells) in the blood, bone marrow and spleen (see Supplemental Figure S2 at http://ajp.amjpathol.org), increased numbers of platelets in the blood (see Supplemental Figure S3 at http://ajp.amjpathol.org), and an enlarged spleen (see Supplemental Figure S4 at http://ajp.amjpathol.org) when compared with their littermate wild-type controls. However, we observed two differences between WBB6F1- KitW-sh/W-sh mice and B6J-KitW-sh/W-sh mice that probably represent effects of strain background: naïve WBB6F1- KitW-sh/W-sh mice, but not naïve B6J-KitW-sh/W-sh mice, exhibited significantly increased numbers of neutrophils (Gr1+, F4/80− cells) in the peritoneal cavity (see Supplemental Figure S5A at http://ajp.amjpathol.org), and B6J-KitW-sh/W-sh mice, but not WBB6F1-KitW-sh/W-sh mice, exhibited significantly reduced numbers of Gr1−, F4/80+ macrophages in the peritoneal cavity (see Supplemental Figure S5B at http://ajp.amjpathol.org), when compared with their age- and gender-matched wild-type controls. In contrast to WBB6F1-KitW/W-v mice, which have reduced numbers of basophils (defined here as c-Kit−CD49b[DX5]+ FcεRI α chain+ cells28) in the blood (see Supplemental Figure S6 at http://ajp.amjpathol.org), we found that numbers of basophils were increased in the blood of B6J-KitW-sh/W-sh mice and WBB6F1-KitW-sh/W-sh mice (see Supplemental Figure S6 at http://ajp.amjpathol.org). However, it is not clear whether basophils have any critical function in the CLP model.

Our results indicate that both strain background, ie, (WB/ReJ × C57BL/6)F1 versus C57BL/6J, and the type of mutation affecting c-Kit expression (ie, KitW/W-v versus KitW-sh/W-sh) can influence the aspects of phenotype we analyzed. Our findings also show that, for most of the phenotypic features assessed in this study, WBB6F1- KitW-sh/W-sh mice more closely resembled C57BL/6J- KitW-sh/W-sh mice than WBB6F1-KitW/W-v mice.

We then investigated the responses of WBB6F1- KitW-sh/W-sh mice to moderately severe and severe CLP. Like the other mast cell-deficient mice analyzed, WBB6F1-KitW-sh/W-sh mice exhibited reduced survival after moderately severe CLP when compared with the corresponding normal mice (58 vs. 6% survival at day 7, respectively, P = 0.001). Engraftment of WBB6F1- KitW-sh/W-sh mice with wild-type BMCMCs improved their survival to levels that were not significantly different from those found in wild-type mice (Figure 4A). By contrast, after severe CLP (Figure 4B), WBB6F1-KitW-sh/W-sh mice exhibited slightly (but not significantly) better survival than either the littermate wild-type controls (30% vs. 17% survival at day 7, respectively, P = 0.21) or the WBB6F1-KitW-sh/W-sh mice that had been engrafted with wild-type mast cells (0% survival at day 7, P = 0.64).

Figure 4.

Figure 4

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Mast cells enhance survival in WBB6F1-KitW-sh/W-sh mice after moderately severe but not severe CLP. A: Survival after moderately severe CLP (50% ligation; single puncture with 22G needle) in wild-type (WBB6F1-Kit+/+) mice (black squares) (n = 12), WBB6F1-KitW-sh/W-sh mast cell-deficient mice (white squares) (n = 16) and WBB6F1-Kit+/+ BMCMCs→WBB6F1-KitW-sh/W-sh mice (blue circles) (n = 9). B: Survival after severe CLP (2/3 ligation; single puncture with 22G needle) in wild-type (WBB6F1-Kit+/+) mice (black squares) (n = 30), WBB6F1-KitW-sh/W-sh mast cell-deficient mice (white squares) (n = 16) and WBB6F1-Kit+/+ BMCMCs→WBB6F1-KitW-sh/W-sh (blue circles) (n = 5). *P < 0.05, ***P < 0.001 versus WBB6F1-KitW-sh/W-sh mice; ††P < 0.01 versus corresponding results for moderately severe CLP. All mice were males that were 12 weeks old at the beginning of the experiment. n.s., not significant. C–E: Numbers of neutrophils (polymorphonuclear leukocytes [PMN]) (C), amount of TNF (D), and numbers of bacterial CFUs (E), in the peritoneal lavage fluid at 18 to 24 hours after severe CLP in wild-type (WBB6F1-Kit+/+) (n = 4 to 13), WBB6F1-KitW-sh/W-sh mast cell-deficient mice (n = 3 to 11) and WBB6F1-Kit+/+BMCMCs→WBB6F1-KitW-sh/W-sh mice (n = 3 to 10). n.s., not significant.

All groups of mice exhibited similar numbers of neutrophils (Figure 4C), amounts of TNF (Figure 4D) and numbers of bacterial CFUs (Figure 4E) in the peritoneal cavity at 18 to 24 hours after severe CLP. However, compared with the corresponding groups of mice on the C57BL/6 background, WBB6F1 wild-type mice and WBB6F1-KitW-sh/W-sh mice engrafted with wild-type mast cells exhibited significantly lower numbers of neutrophils (compare Figures 3B and 4C), amounts of TNF (compare Figures 3C and 4D) and bacterial CFUs (compare Figures 3D and 4E) in the peritoneal cavity at 18 to 24 hours after severe CLP. These observations support the conclusion that C57BL/6 mice exhibit a more pronounced inflammatory response to severe CLP than do WBB6F1 mice, at least as judged by levels of TNF and neutrophils in the peritoneal cavity. The differences in the findings in severe CLP in wild-type C57BL/6 vs. WBB6F1 mice also are consistent with the possibility that strain differences affecting mast cells result in mast cells having a more substantial effect in enhancing inflammation, and reducing survival, after severe CLP in C57BL/6 mice than in the WBB6F1 animals.

Mast Cell-Derived TNF Can Contribute to Bacterial Proliferation and Hasten Death during Infection with S. typhimurium

Our findings in mice that were subjected to severe CLP were unexpected, since prior work indicated that mast cells can have protective effects in moderately severe CLP and in other models of bacterial infection.1,2,3,4,5,7,8,9,10,11,13 We therefore decided to examine the role of mast cells and mast cell-derived TNF in another model of severe bacterial infection.

S. typhimurium is a Gram-negative intracellular pathogen that causes a deadly systemic infection in mice that resembles typhoid fever in humans.34 We induced a severe infection with S. typhimurium in mice by injecting a virulent strain (SL1344) of S. typhimurium (SL1344) into the peritoneal cavity.35 In contrast to mast cell-deficient WBB6F1-KitW/W-v mice (which we previously showed died more rapidly after i.p. inoculation with S. typhimurium than did the corresponding wild-type mice27), mast cell-deficient C57BL/6-KitW-sh/W-sh mice exhibited slightly but significantly (P = 0.019) prolonged survival, compared with the corresponding wild-type mice, on i.p. injection of bioluminescent S. typhimurium (Figure 5A).

Figure 5.

Figure 5

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Mast cells impair innate immunity in C57BL/6-KitW-sh/W-sh mice during infection with S. typhimurium. (A and B) Survival and (C), bioluminescent signals at 5 days in mice that received 25 CFU S. typhimurium SL1344lux via an i.p. injection; wild-type C57BL/6J mice (B6-Kit+/+, black squares) (n = 18), mast cell-deficient C57BL/6-KitW-sh/W-sh mice (B6-KitW-sh/W-sh, white squares, the same data are shown in A and B for clarity) (n = 21), wild-type B6-Tnf+/+ BMCMCs→ B6-KitW-sh/W-sh mice (blue circles) (n = 26) and B6-Tnf−/− BMCMCs→B6-KitW-sh/W-sh mice (yellow rhomboids) (n = 14). In C, the intensity of bioluminescence is depicted using a pseudocolor scale superimposed over a grayscale reference image of the mice. Results in (C) are representative of the similar results obtained in 3 to 4 separate experiments. In (A), *P < 0.05 versus B6 mice; in (B), *P < 0.05, ***P < 0.0001 versus B6-Tnf+/+ BMCMCs->B6-KitW-sh/W-sh mice. All mice were males that were 12 weeks old at the beginning of the experiment.

We then assessed whether adoptive transfer of mast cells influenced the survival of C57BL/6-KitW-sh/W-sh mice in this model. Like after severe CLP (Figure 3A), we found that C57BL/6-KitW-sh/W-sh mice selectively engrafted with wild-type (C57BL/6-Tnf+/+) mast cells had significantly shorter survival after S. typhimurium inoculation than either mast cell-deficient C57BL/6-KitW-sh/W-sh mice or C57BL/6-KitW-sh/W-sh mice engrafted with Tnf−/− BMCMCs (Figure 5B). Tnf+/+ BMCMCs→KitW-sh/W-sh mice also exhibited stronger bioluminescent signals on d 5 of infection, reflecting greater numbers of bioluminescent S. typhimurium, than did either mast cell-deficient C57BL/6-KitW-sh/W-sh mice or C57BL/6-KitW-sh/W-sh mice engrafted with Tnf−/− BMCMCs (Figure 5C).

However, when assessed at 18 to 24 hours after S. typhimurium inoculation, C57BL/6-KitW-sh/W-sh mice engrafted with wild-type mast cells exhibited significantly lower numbers of neutrophils in the peritoneal cavity than either mast cell-deficient C57BL/6-KitW-sh/W-sh mice or C57BL/6-KitW-sh/W-sh mice engrafted with Tnf−/− mast cells (Supplemental Figure S7 at http://ajp.amjpathol.org). This finding is the opposite of that obtained when these groups of mice were subjected to the severe model of CLP (see Figure 3B). Moreover, when we analyzed the same mice shown in Supplemental Figure S7 (http://ajp.amjpathol.org) for intraperitoneal levels of TNF, we found that no TNF was detectable in any of the mice at 18 to 24 hours after S. typhimurium inoculation. Thus, this model of S. typhimurium infection is associated with substantially lower levels of intraperitoneal neutrophils, and TNF production, than our models of CLP.

These results show that TNF production by mast cells can enhance bacterial proliferation (as reflected by increased bioluminescent signals [Figure 5C]) and hasten death (Figure 5B) in this model of severe infection with S. typhimurium in C57BL/6-KitW-sh/W-sh mice. However, unlike in our model of severe CLP, in mice infected with S. typhimurium, the hastened death observed in C57BL/6- KitW-sh/W-sh mice containing wild-type mast cells was not accompanied by the presence of higher numbers of peritoneal neutrophils than in C57BL/6-KitW-sh/W-sh mice containing Tnf−/− mast cells (Supplemental Figure S7 at http://ajp.amjpathol.org).

Discussion

Our findings show that mast cell engraftment into the peritoneal cavity of mast cell-deficient mice can have effects that range from enhancing or impairing the animals’ innate immunity to bacterial infection, with the result obtained in individual experiments depending on both the details of the model of infection analyzed and the type of mast cell-deficient mice studied. Moreover, we show in C57BL/6-KitW-sh/W-sh mice that mast cell-derived TNF is not required for mast cells to enhance survival in one model of innate immunity, moderately severe CLP, but that mast cell-derived TNF can contribute to the deleterious effects of mast cells in severe CLP or after intraperitoneal inoculation with S. typhimurium.

We investigated CLP both because this model is widely used (and has been called the “gold standard”) in sepsis research,36 and because many studies investigating the potential roles of mast cells in innate immunity to bacterial infection have used this model.1,3,4,5,6,8,10,13 However, the great majority of such studies have investigated moderately severe CLP,1,3,4,5,6,8,10 in which most of the wild-type mice survive. In the present study, we used three different types of mast cell-engrafted, genetically mast cell-deficient mice to reinvestigate the contribution of mast cells to survival after moderately severe CLP and to analyze the role of mast cells in severe CLP, in which most wild-type mice succumb.

It has been reported that mast cells can enhance survival in WBB6F1-KitW/W-v mice subjected to moderately severe CLP,1,3,4,5,6,8,10,13 and we have confirmed that finding (Figure 1A). Moreover, we report herein that mast cells also can enhance survival after moderately severe CLP in C57BL/6-KitW-sh/W-sh mice (Figure 2A) and in WBB6F1-KitW-sh/W-sh mice (Figure 4A). These findings indicate that the beneficial function of mast cells on survival in this model of innate immunity to bacterial infection can be observed in mice of two different strain backgrounds (ie, WBB6F1 and C57BL/6) and in WBB6F1 mice with either of two distinct mutations, which result in mast cell deficiency (ie, KitW/W-v and KitW-sh/W-sh). However, our data indicate that, during severe CLP, mast cells either can have no statistically significant effect on survival (in mast cell-engrafted WBB6F1-KitW/W-v [Figure 1B] or WBB6F1-KitW-sh/W-sh [Figure 4B] mice) or can actually impair survival (in C57BL/6-KitW-sh/W-sh mice engrafted with wild-type mast cells [Figure 3A]).

Our findings thus show that variation in mortality rates that are observed among different types of mast cell-engrafted mast cell-deficient mice subjected to severe CLP can reflect differences in their strain background, as well as differences in the consequences of the mutations that result in their mast cell deficiency. However, it bears emphasis that WBB6F1-KitW/W-v and C57BL/6-KitW-sh/W-sh mice,14,15,16,17,18,19,20,21 as well as the WBB6F1-KitW-sh/W-sh mice that were generated and characterized in the present study, exhibit a variety of consequences of their mutations in addition to a profound deficiency in mast cells. These additional abnormalities clearly must be kept in mind when interpreting the results of experiments that employ such animals to investigate mast cell functions.

For example, the phenotypic abnormalities of WBB6F1-KitW/W-v, C57BL/6-KitW-sh/W-sh, and WBB6F1- KitW-sh/W-sh mice include alterations in the numbers of hematopoietic populations other than mast cells, including neutrophils14,15,16 (Supplemental Figure S1 at http://ajp.amjpathol.org), macrophages16 (Supplemental Figure S4B at http://ajp.amjpathol.org), and basophils (Supplemental Figure S5 at http://ajp.amjpathol.org). Therefore, it is possible that in some biological responses the presence of adoptively transferred mast cells in genetically mast cell-deficient mice can compensate for some of the consequences of the other defects in such animals. Indeed, this has been proposed as one explanation of why some models of experimental arthritis that feature an important role for neutrophils are poorly expressed in the relatively neutrophil-deficient WBB6F1-KitW/W-v mice (unless the animals contain adoptively transferred populations of mast cells), but are normally expressed in mast cell-deficient C57BL/6-KitW-sh/W-sh mice, which have levels of neutrophils higher than those in the corresponding wild-type mice.15,16

Compensation by adoptively transferred mast cells for some consequence of the animal’s relative deficiency in neutrophils may also contribute to the beneficial role of mast cells observed in mast cell-engrafted WBB6F1- KitW/W-v mice subjected to moderately severe CLP. However, we showed that adoptive transfer of mast cells also increases survival after moderately severe CLP in both C57BL/6-KitW-sh/W-sh and WBB6F1-KitW-sh/W-sh mice, which have baseline levels of neutrophils in the blood, bone marrow, spleen, and peritoneal cavity that are not reduced and in many sites are significantly increased compared with those in the corresponding wild-type mice. Taken together, this evidence shows that, in C57BL/6-KitW-sh/W-sh and WBB6F1-KitW-sh/W-sh mice, mast cells must contribute to improved survival after moderately severe CLP by mechanisms beyond simply compensating for a defect in numbers of neutrophils.

Our studies in C57BL/6-KitW-sh/W-sh mice engrafted with wild-type versus TNF-deficient mast cells indicated that wild-type mast cells can impair survival in C57BL/6-KitW-sh/W-sh mice subjected to severe CLP, and that this effect is significantly reduced if the adoptively transferred mast cell population lacks the ability to make TNF (Figure 3A). It is likely that death after CLP reflects the operation of complex processes, and additional work will be required to understand how mast cell-derived TNF impairs survival in this setting. One possibility is that the ability of the adoptively transferred wild-type mast cells to produce TNF and other mediators, when added to the already increased levels of neutrophils at baseline in C57BL/6-KitW-sh/W-sh mice, may result in an environment that in some ways may be more “pro-inflammatory” than that present in the corresponding wild-type animals. However, we found that the adoptive transfer of wild-type mast cells to C57BL/6-KitW-sh/W-sh mice appeared to allow these animals to respond to severe CLP, at least with respect to intraperitoneal levels of TNF and neutrophils, in a way that was very similar to that of the corresponding wild-type mice. Thus, even though C57BL/6-KitW-sh/W-sh mice had significantly more neutrophils in the blood, bone marrow and spleen at baseline than did the corresponding wild-type mice (Supplemental Figure 2), when tested 18 to 24 hours after the induction of moderately severe or severe CLP, the levels of intraperitoneal neutrophils or TNF were quite similar in C57BL/6-KitW-sh/W-sh mice that had been engrafted with wild-type mast cells and in the corresponding wild-type mice (Figures 2, B and C, and 3, B and C).

It has been proposed that neutrophils can express opposing roles during CLP and other innate immune response to bacterial infection, promoting bacterial clearance but, in severe infections, also contributing to organ damage.37 However, C57BL/6-KitW-sh/W-sh mice engrafted with wild-type versus TNF-deficient mast cells may not differ only in the levels of TNF (Figure 2C) and neutrophils (Figure 2B) observed after severe CLP, but also in other mediators produced in this setting, which may be influenced by TNF and/or neutrophils. Proving which of these factors contribute most to lethality in this setting may be difficult. For example, using approaches to block the activity of TNF (which has been done by others in studies of moderately severe CLP)1,38 would block TNF from all sources, not just TNF of mast cell origin.

To investigate the possibility that neutrophils contribute to mortality in our model of severe CLP, we attempted to diminish neutrophils in C57BL/6J mice using both the anti-Ly-6G/Ly-6C antibody (clone RB6-8C5)39 and the anti-Ly-6G antibody (clone 1A8).40 However, in our hands, treatment of mice with either of these antibodies, which have been used to reduce neutrophils in mice in vivo,37,41 also resulted in a less dramatic, but significant, reduction in blood monocytes after severe CLP (data not shown). Moreover, we found that the administration of an isotype-matched control antibody (ie, rat IgG2a, κ, clone R35-95, catalog number 553927, BD Biosciences) to wild-type C57BL/6J mice also resulted in a significant drop in blood neutrophils, as well as a significant reduction in the mortality associated with severe CLP (data not shown). Thus, our attempts to achieve a selective and specific reduction in neutrophils in our model of severe CLP have not yet yielded readily interpretable results. In summary, although we can conclude that the presence of wild-type (as opposed to TNF-deficient) mast cells in C57BL/6-KitW-sh/W-sh mice subjected to severe CLP results in these animals developing a more “pro-inflammatory” response to the infection, as judged by levels of intraperitoneal TNF and neutrophils, it is not yet clear to what extent these findings may contribute to the ability of wild-type mast cells to increase the mortality of severe CLP.

In light of the provocative finding that mast cell-derived TNF increased mortality in severe CLP in C57BL/6- KitW-sh/W-sh mice, we re-examined the hypothesis that mast cells can contribute to survival during moderately severe CLP in part through the production of TNF.1 We found that, in C57BL/6-KitW-sh/W-sh mice, mast cell-derived TNF did not detectably contribute to the process by which mast cells enhanced survival (Figure 2A). At 18 to 24 hours after the induction of moderately severe CLP, intraperitoneal levels of neutrophils were substantially higher in C57BL/6-Kit+/+ wild-type mice or in C57BL/6- KitW-sh/W-sh mice engrafted with wild-type or TNF-deficient mast cells than they were in mast cell-deficient C57BL/6-KitW-sh/W-sh mice (Figure 2B), and the numbers of bacterial CFUs were significantly higher in the mast cell-deficient C57BL/6-KitW-sh/W-sh mice than in each of the other three groups (Figure 2D). However, at 18 to 24 hours after CLP, all four of these groups had very similar levels of intraperitoneal TNF (Figure 2C). C57BL/6-KitW-sh/W-sh mice and the corresponding wild-type mice also exhibited similar levels of intraperitoneal TNF and neutrophils 6 hours after induction of severe CLP (Supplemental Figure S1 at http://ajp.amjpathol.org). Taken together with the survival data, which showed that only the survival of mast cell-deficient C57BL/6- KitW-sh/W-sh mice was significantly worse than that of the C57BL/6-Kit+/+ wild-type mice (Figure 2A), these findings argue against a critical role for mast cell-derived TNF either in altering intraperitoneal levels of TNF or in contributing to the mast cell-dependent increases in intraperitoneal neutrophils or decreases in intraperitoneal bacterial CFUs during moderately severe CLP. Mast cells also have been reported not to influence the increased levels of intraperitoneal TNF which were observed in mice after they received an intraperitoneal injection with a thousand Klebsiella pneumonia bacteria.12

We do not consider it surprising that mast cells can contribute to survival after moderately severe CLP even if they lack the ability to make TNF. Indeed, there is evidence that several mast cell products can enhance survival during innate immune responses to bacterial infection. In studies of moderately severe CLP, it has been reported that mast cells can enhance survival by production of interleukin-642 or by the action of mast cell-associated proteases including mast cell carboxypeptidase A or neurolysin (which can enhance survival by degrading potentially toxic endogenous peptides produced during CLP, such as endothelin-18,13 or neurolysin13). In studies of mice injected intraperitoneally with K. pneumoniae, survival can be enhanced by mouse mast cell protease 6.12 Thus, analyses of moderately severe CLP and other models of bacterial infection show that mast cells can contribute to survival in such settings via several different mechanisms. Moreover, although work by several groups (including our own) has shown that TNF also can confer benefit during CLP4,38 or other models of innate immune responses to bacteria43,44 in mice, it is well known that mast cells represent just one among many potential cellular sources of TNF.45,46. Thus, while we haven’t ruled out an important role for mast cell-derived TNF in other models of innate immunity, our present study shows that, at least in C57BL/6 mice, mast cells are unlikely to represent a critical source of TNF during moderately severe CLP.

To extend our observations beyond models of CLP, we investigated a different model of severe bacterial infection, that induced by the intraperitoneal injection of S. typhimurium.27 In C57BL/6-KitW-sh/W-sh mice, we found that mice engrafted with wild-type mast cells died more rapidly than those engrafted with TNF-deficient mast cells or those which virtually lacked mast cells (Figure 5). This result indicated that mast cell-derived TNF can exacerbate the pathology associated with this model of severe infection, as it did in severe CLP. However, this model of S. typhimurium infection was associated with substantially lower numbers of intraperitoneal neutrophils at 18 to 24 hours after the initiation of infection than the model of severe CLP (compare Figures 3B and Supplemental Figure S7 at http://ajp.amjpathol.org). Moreover, numbers of neutrophils at 18 to 24 hours after injection of S. typhimurium were significantly lower in C57BL/6-KitW-sh/W-sh mice engrafted with wild-type mast cells than in either C57BL/6-KitW-sh/W-sh mice or C57BL/6-KitW-sh/W-sh mice engrafted with TNF-deficient mast cells (Supplemental Figure S7 at http://ajp.amjpathol.org), and levels of intraperitoneal TNF were below the limits of detection in each of these groups.

Our findings show that mast cell-derived TNF can increase and/or hasten mortality in C57BL/6-KitW-sh/W-sh mice in two very different models of severe bacterial infection: severe CLP and intraperitoneal inoculation with S. typhimurium. Our findings also suggest that the detrimental effect of mast cell-derived TNF in innate immunity to bacterial infection can be observed in settings that are or are not associated with mast cell-derived TNF-dependent enhancement of the numbers of neutrophils, or levels of TNF, in the peritoneal cavity of the infected mice.

In summary, our findings show that the roles of mast cells, and mast cell-derived TNF, in innate immune responses to bacterial infection depend on the model of infection analyzed, and on the type of genetically mast cell-deficient mice used to investigate such models. In all three types of mast cell-deficient mice analyzed, mast cells significantly improved survival in mice subjected to moderately severe CLP. In C57BL/6-KitW-sh/W-sh mice, mast cells also increased levels of intraperitoneal neutrophils in this setting and improved bacterial clearance, but these effects were seen in C57BL/6-KitW-sh/W-sh mice that contained either wild-type or TNF-deficient mast cells. Our findings in the moderately severe model of CLP thus are consistent with the now widely held view that when mast cells contribute to effective innate immune responses to infection, they do so at least in part by enhancing neutrophil responses and bacterial clearance.1,2,12,31,32 However, we also showed that mast cells can mediate such effects in moderately severe CLP even if they are unable to produce TNF. By contrast, we found that mast cells, and mast cell-derived TNF, can increase and/or hasten mortality in C57BL/6-KitW-sh/W-sh mice in two different models of severe bacterial infection: severe CLP and intraperitoneal inoculation with S. typhimurium. While the mechanisms by which mast cell-derived TNF can increase pathology in these two models remain to be defined, at 18 to 24 hours after the onset of infection, mast cell-derived TNF increased numbers of intraperitoneal neutrophils (as well as intraperitoneal levels of TNF) in the severe CLP model, but not during infection with S. typhimurium. These results raise the possibility that mast cell-derived TNF can have deleterious effects in these two models via distinct mechanisms of action.

Acknowledgments

We thank Justin Kenkel for technical assistance.

Footnotes

Address reprint requests to Stephen J. Galli, M.D., or Adrian M. Piliponsky, Ph.D., Stanford University School of Medicine, Pathology, L-235, 300 Pasteur Drive, Stanford, CA 94305-5324. E-mail: sgalli@stanford.edu or adrianp@stanford.edu.

Supported by United States Public Health Service grants (to S.J.G.) AI23990, AI070813, and CA72074.

S.J.G. occasionally consults for companies that sell and/or are developing agents to treat allergic disorders, including Amgen Inc., Genentech, and Novartis. S.J.G. is a member of the Scientific Advisory Board of Tunitas Therapeutics, which is developing protein therapeutics for the treatment of allergic diseases.

Supplemental material for this article can be found on http://ajp.amjpathol.org.

References

  1. Echtenacher B, Mannel DN, Hultner L. Critical protective role of mast cells in a model of acute septic peritonitis. Nature. 1996;381:75–77. doi: 10.1038/381075a0. [DOI] [PubMed] [Google Scholar]
  2. Malaviya R, Ikeda T, Ross E, Abraham SN. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature. 1996;381:77–80. doi: 10.1038/381077a0. [DOI] [PubMed] [Google Scholar]
  3. Prodeus AP, Zhou X, Maurer M, Galli SJ, Carroll MC. Impaired mast cell-dependent natural immunity in complement C3-deficient mice. Nature. 1997;390:172–175. doi: 10.1038/36586. [DOI] [PubMed] [Google Scholar]
  4. Maurer M, Echtenacher B, Hultner L, Kollias G, Mannel DN, Langley KE, Galli SJ. The c-kit ligand, stem cell factor, can enhance innate immunity through effects on mast cells. J Exp Med. 1998;188:2343–2348. doi: 10.1084/jem.188.12.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Supajatura V, Ushio H, Nakao A, Akira S, Okumura K, Ra C, Ogawa H. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J Clin Invest. 2002;109:1351–1359. doi: 10.1172/JCI14704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jippo T, Morii E, Ito A, Kitamura Y. Effect of anatomical distribution of mast cells on their defense function against bacterial infections: demonstration using partially mast cell-deficient tg/tg mice. J Exp Med. 2003;197:1417–1425. doi: 10.1084/jem.20022157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. McLachlan JB, Hart JP, Pizzo SV, Shelburne CP, Staats HF, Gunn MD, Abraham SN. Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection. Nat Immunol. 2003;4:1199–1205. doi: 10.1038/ni1005. [DOI] [PubMed] [Google Scholar]
  8. Maurer M, Wedemeyer J, Metz M, Piliponsky AM, Weller K, Chatterjea D, Clouthier DE, Yanagisawa MM, Tsai M, Galli SJ. Mast cells promote homeostasis by limiting endothelin-1-induced toxicity. Nature. 2004;432:512–516. doi: 10.1038/nature03085. [DOI] [PubMed] [Google Scholar]
  9. Xu X, Zhang D, Lyubynska N, Wolters PJ, Killeen NP, Baluk P, McDonald DM, Hawgood S, Caughey GH. Mast cells protect mice from Mycoplasma pneumonia. Am J Respir Crit Care Med. 2006;173:219–225. doi: 10.1164/rccm.200507-1034OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Orinska Z, Maurer M, Mirghomizadeh F, Bulanova E, Metz M, Nashkevich N, Schiemann F, Schulmistrat J, Budagian V, Giron-Michel J, Brandt E, Paus R, Bulfone-Paus S. IL-15 constrains mast cell-dependent antibacterial defenses by suppressing chymase activities. Nat Med. 2007;13:927–934. doi: 10.1038/nm1615. [DOI] [PubMed] [Google Scholar]
  11. Siebenhaar F, Syska W, Weller K, Magerl M, Zuberbier T, Metz M, Maurer M. Control of Pseudomonas aeruginosa skin infections in mice is mast cell-dependent. Am J Pathol. 2007;170:1910–1916. doi: 10.2353/ajpath.2007.060770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Thakurdas SM, Melicoff E, Sansores-Garcia L, Moreira DC, Petrova Y, Stevens RL, Adachi R. The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections. J Biol Chem. 2007;282:20809–20815. doi: 10.1074/jbc.M611842200. [DOI] [PubMed] [Google Scholar]
  13. Piliponsky AM, Chen CC, Nishimura T, Metz M, Rios EJ, Dobner PR, Wada E, Wada K, Zacharias S, Mohanasundaram UM, Faix JD, Abrink M, Pejler G, Pearl RG, Tsai M, Galli SJ. Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis. Nat Med. 2008;14:392–398. doi: 10.1038/nm1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chervenick PA, Boggs DR. Decreased neutrophils and megakaryocytes in anemic mice of genotype W/W. J Cell Physiol. 1969;73:25–30. doi: 10.1002/jcp.1040730104. [DOI] [PubMed] [Google Scholar]
  15. Zhou JS, Xing W, Friend DS, Austen KF, Katz HR. Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis. J Exp Med. 2007;204:2797–2802. doi: 10.1084/jem.20071391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nigrovic PA, Gray DH, Jones T, Hallgren J, Kuo FC, Chaletzky B, Gurish M, Mathis D, Benoist C, Lee DM. Genetic inversion in mast cell-deficient (W(sh)) mice interrupts corin and manifests as hematopoietic and cardiac aberrancy. Am J Pathol. 2008;173:1693–1701. doi: 10.2353/ajpath.2008.080407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grimbaldeston MA, Chen CC, Piliponsky AM, Tsai M, Tam SY, Galli SJ. Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo. Am J Pathol. 2005;167:835–848. doi: 10.1016/S0002-9440(10)62055-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tono T, Tsujimura T, Koshimizu U, Kasugai T, Adachi S, Isozaki K, Nishikawa S, Morimoto M, Nishimune Y, Nomura S. c-kit Gene was not transcribed in cultured mast cells of mast cell-deficient Wsh/Wsh mice that have a normal number of erythrocytes and a normal c-kit coding region. Blood. 1992;80:1448–1453. [PubMed] [Google Scholar]
  19. Wolters PJ, Mallen-St Clair J, Lewis CC, Villalta SA, Baluk P, Erle DJ, Caughey GH. Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient Kit(W-sh)/Kit(W-sh) sash mice. Clin Exp Allergy. 2005;535:82–88. doi: 10.1111/j.1365-2222.2005.02136.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kitamura Y, Go S, Hatanaka K. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood. 1978;52:447–452. [PubMed] [Google Scholar]
  21. Galli SJ, Kitamura Y. Genetically mast-cell-deficient W/Wv and Sl/Sld mice. Their value for the analysis of the roles of mast cells in biologic responses in vivo. Am J Pathol. 1987;127:191–198. [PMC free article] [PubMed] [Google Scholar]
  22. Lyon MF, Glenister PH. A new allele sash (Wsh) at the W-locus and a spontaneous recessive lethal in mice. Genet Res. 1982;39:315–322. doi: 10.1017/s001667230002098x. [DOI] [PubMed] [Google Scholar]
  23. Stevens J, Loutit JF. Mast cells in spotted mutant mice (W Ph mi). Proc R Soc Lond B Biol Sci. 1982;215:405–409. doi: 10.1098/rspb.1982.0050. [DOI] [PubMed] [Google Scholar]
  24. Duttlinger R, Manova K, Chu TY, Gyssler C, Zelenetz AD, Bachvarova RF, Besmer P. W-sash affects positive and negative elements controlling c-kit expression: ectopic c-kit expression at sites of kit-ligand expression affects melanogenesis. Development. 1993;118:705–717. doi: 10.1242/dev.118.3.705. [DOI] [PubMed] [Google Scholar]
  25. Korner H, Cook M, Riminton DS, Lemckert FA, Hoek RM, Ledermann B, Kontgen F, Fazekas de St Groth B, Sedgwick JD. Distinct roles for lymphotoxin-alpha and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue. Eur J Immunol. 1997;27:2600–2609. doi: 10.1002/eji.1830271020. [DOI] [PubMed] [Google Scholar]
  26. Burns-Guydish SM, Olomu IN, Zhao H, Wong RJ, Stevenson DK, Contag CH. Monitoring age-related susceptibility of young mice to oral Salmonella enterica serovar Typhimurium infection using an in vivo murine model. Pediatr Res. 2005;58:153–158. doi: 10.1203/01.PDR.0000157725.44213.C4. [DOI] [PubMed] [Google Scholar]
  27. Chatterjea D, Burns-Guydish SM, Sciuto TE, Dvorak A, Contag CH, Galli SJ. Adoptive transfer of mast cells does not enhance the impaired survival of Kit(W)/Kit(W-v) mice in a model of low dose intraperitoneal infection with bioluminescent Salmonella typhimurium. Immunol Lett. 2005;99:122–129. doi: 10.1016/j.imlet.2005.02.015. [DOI] [PubMed] [Google Scholar]
  28. Mukai K, Matsuoka K, Taya C, Suzuki H, Yokozeki H, Nishioka K, Hirokawa K, Etori M, Yamashita M, Kubota T, Minegishi Y, Yonekawa H, Karasuyama H. Basophils play a critical role in the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells. Immunity. 2005;23:191–202. doi: 10.1016/j.immuni.2005.06.011. [DOI] [PubMed] [Google Scholar]
  29. Nakae S, Suto H, Iikura M, Kakurai M, Sedgwick JD, Tsai M, Galli SJ. Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF. J Immunol. 2006;176:2238–2248. doi: 10.4049/jimmunol.176.4.2238. [DOI] [PubMed] [Google Scholar]
  30. Yamashita Y, Charles N, Furumoto Y, Odom S, Yamashita T, Gilfillan AM, Constant S, Bower MA, Ryan JJ, Rivera J. Cutting edge: genetic variation influences Fc epsilonRI-induced mast cell activation and allergic responses. J Immunol. 2007;179:740–743. doi: 10.4049/jimmunol.179.2.740. [DOI] [PubMed] [Google Scholar]
  31. Malaviya R, Abraham SN. Mast cell modulation of immune responses to bacteria. Immunol Rev. 2001;179:16–24. doi: 10.1034/j.1600-065x.2001.790102.x. [DOI] [PubMed] [Google Scholar]
  32. Dawicki W, Marshall JS. New and emerging roles for mast cells in host defence. Curr Opin Immunol. 2007;19:31–38. doi: 10.1016/j.coi.2006.11.006. [DOI] [PubMed] [Google Scholar]
  33. Zhang Y, Ramos BF, Jakschik BA. Neutrophil recruitment by tumor necrosis factor from mast cells in immune complex peritonitis. Science. 1992;258:1957–1959. doi: 10.1126/science.1470922. [DOI] [PubMed] [Google Scholar]
  34. Monack DM, Mueller A, Falkow S. Persistent bacterial infections: the interface of the pathogen and the host immune system. Nat Rev Microbiol. 2004;2:747–765. doi: 10.1038/nrmicro955. [DOI] [PubMed] [Google Scholar]
  35. Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature. 1981;291:238–239. doi: 10.1038/291238a0. [DOI] [PubMed] [Google Scholar]
  36. Rittirsch D, Hoesel LM, Ward PA. The disconnect between animal models of sepsis and human sepsis. J Leukoc Biol. 2007;81:137–143. doi: 10.1189/jlb.0806542. [DOI] [PubMed] [Google Scholar]
  37. Hoesel LM, Neff TA, Neff SB, Younger JG, Olle EW, Gao H, Pianko MJ, Bernacki KD, Sarma JV, Ward PA. Harmful and protective roles of neutrophils in sepsis. Shock. 2005;24:40–47. doi: 10.1097/01.shk.0000170353.80318.d5. [DOI] [PubMed] [Google Scholar]
  38. Echtenacher B, Falk W, Mannel DN, Krammer PH. Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis. J Immunol. 1990;145:3762–3766. [PubMed] [Google Scholar]
  39. Brummer E, Sugar AM, Stevens DA. Immunological activation of polymorphonuclear neutrophils for fungal killing: studies with murine cells and blastomyces dermatitidis in vitro. J Leukoc Biol. 1984;36:505–520. doi: 10.1002/jlb.36.4.505. [DOI] [PubMed] [Google Scholar]
  40. Fleming TJ, Fleming ML, Malek TR. Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family, J Immunol. 1993;151:2399–2408. [PubMed] [Google Scholar]
  41. Daley JM, Thomay AA, Connolly MD, Reichner JS, Albina JE. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol. 2008;83:64–70. doi: 10.1189/jlb.0407247. [DOI] [PubMed] [Google Scholar]
  42. Sutherland RE, Olsen JS, McKinstry A, Villalta SA, Wolters PJ. Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing. J Immunol. 2008;181:5598–5605. doi: 10.4049/jimmunol.181.8.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med. 1996;184:1397–1411. doi: 10.1084/jem.184.4.1397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Marino MW, Dunn A, Grail D, Inglese M, Noguchi Y, Richards E, Jungbluth A, Wada H, Moore M, Williamson B, Basu S, Old LJ. Characterization of tumor necrosis factor-deficient mice. Proc Natl Acad Sci USA. 1997;94:8093–8098. doi: 10.1073/pnas.94.15.8093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mannel DN, Echtenacher B. TNF in the inflammatory response. Chem Immunol. 2000;74:141–161. [PubMed] [Google Scholar]
  46. Beutler BA. The role of tumor necrosis factor in health and disease. J Rheumatol Suppl. 1999;57:16–21. [PubMed] [Google Scholar]

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