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Keywords:

  • Complement;
  • Inflammation;
  • Mast cells;
  • Neutrophil;
  • Toll-like receptors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

The in vitro macrophage response to zymosan has been attributed to Toll-like receptor 2 (TLR2). Whether TLR2 is obligatory for the zymosan-induced in vivo response has not been assessed. The importance of this question is underscored by the fact that zymosan activates complement in a cell-independent manner. We have investigated whether the in vitro observation of TLR2 as the dominant zymosan receptor on macrophages would translate to an experimental peritonitis model in vivo. We have treated mice with zymosan, resulting in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. Zymosan-mediated leukocyte recruitment was TLR2 independent, but was predominantly dependent on the complement components, C3 and C5a with a minor contribution from LTB4. Peritoneal neutrophilia was 50% mast cell dependent and this defect was reproduced using C5a receptor (C5aR)-deficient mast cells in mast cell-deficient mice, suggesting that C5aR is responsible for mast cell activation following zymosan challenge. By 24 h, the response to zymosan involved primarily monocyte recruitment and was C3 and C5aR independent. Taken together, these studies indicate that the in vivo inflammatory response to zymosan does not necessarily mimic the TLR2 dependence observed in vitro, and that complement plays a dominant role in early, but not late, zymosan-mediated peritonitis.

Abbreviations:
BMMC:

bone marrow-derived mast cells

C5aR:

C5a receptor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Toll-like receptors (TLR) are germline encoded, evolutionarily conserved pattern-recognition receptors. Activation of TLR, via recognition of pathogen-associated molecular patterns, triggers the host defense system, resulting in the initiation of an innate immune response 13. TLR signal via their Toll/IL-1 receptor domains 2, and the ligation of many TLR, including TLR2, results in the recruitment of the adaptor protein myeloid differentiation factor 88 (MyD88) followed by the activation of NF-κB and MAPK. This results in the production of various cytokines, chemokines and other pro-inflammatory molecules that facilitate leukocyte recruitment and clearance of infection.

TLR2 has been identified as the receptor responsible for immunorecognition of the yeast cell-wall particle, zymosan. Indeed, Ozinsky et al.4 and Underhill et al. 5 have shown that TLR2 is recruited to zymosan-containing phagosomes and is required for the inflammatory response in various isolated cell lines. Zymosan has also been used in vivo, in models of both arthritis and pleurisy 6, 7. Takeshita et al.6 observed a reduction in zymosan-induced pleural neutrophilia when mice were pre-treated with a blocking mAb against TLR2. Likewise, Frasnelli et al.7, using a model of zymosan-induced arthritis, observed reduced synovial joint inflammation in TLR2-deficient mice compared with wild-type controls. It is worth noting, however, that zymosan-mediated immune responses were not completely abrogated by the absence of functional TLR2, suggesting that additional receptors may contribute to in vivo zymosan recognition.

Indeed, additional receptors and mediators have been implicated in zymosan-mediated inflammation, particularly dectin-1, complement and the leukotriene B4 (LTB4) 813. Dectin-1 has been shown to mediate the nonopsonic in vitro recognition of zymosan 14 and collaborates with TLR2 in the induction of pro-inflammatory cytokine production 15, 16, although its expression appears to be primarily limited to myeloid cells 17. Furthermore, LTB4 signaling has been demonstrated to play a role in mediating leukocyte recruitment in murine models of zymosan-induced peritonitis 12, 13. Other studies have shown that zymosan can activate plasma in a cell-independent and thus, presumably TLR2- and dectin-1-independent manner, to generate pro-inflammatory complement components 10, 11, 18. Indeed, Colten's group 19, 20 has shown that co-culture of zymosan with normal human sera results in the release of histaminase and β-glucuronidase from neutrophils; an effect abrogated when zymosan was co-cultured with heat-inactivated sera 19 or sera from C3-deficient individuals 20. These data suggest potentially important TLR2-independent, complement- and leukotriene-dependent pathways of zymosan recognition.

We have systematically assessed zymosan-induced peritonitis, to understand the roles of TLR2, LTB4 and complement in in vivo zymosan detection and ensuing leukocyte recruitment, a hallmark feature of the inflammatory response. Our results clearly demonstrate that zymosan induces an inflammatory response that is largely TLR2 independent at both early and late time points. Indeed, we demonstrate that the presence of an intact complement pathway is of much greater importance in mediating the early in vivo peritoneal neutrophil response to zymosan. Finally, we show that mast cells, known to play an important role in peritonitis, are activated via C5a receptor (C5aR) following zymosan administration.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Zymosan induces peritonitis

We have treated mice with increasing doses of zymosan (i.p.) and, after 4 h, assessed circulating leukocyte counts, as well as leukocyte recruitment into both the peritoneum and lung. Treatment of wild-type mice with zymosan did not induce a significant change in circulating leukocyte counts (Fig. 1A). By contrast, the number of leukocytes in the peritoneal exudate was significantly increased 4 h following challenge with increasing doses of zymosan (Fig. 1B). We have previously shown that administration of LPS into the peritoneal cavity causes profound neutrophil recruitment into lungs 21. By contrast, treatment of mice with zymosan (5 mg/kg, i.p. × 4 h) did not result in significant neutrophil trapping in the lung (Fig. 1C). Clearly, zymosan did not cause obvious systemic leukocyte activation (drop or increase in circulating counts, or neutrophilia in the lung), but avidly recruited neutrophils into the peritoneal cavity.

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Figure 1. Effect of zymosan on circulating leukocyte counts and leukocyte accumulation in the peritoneum and lung. C57BL/6 mice were challenged i.p. with saline or zymosan (Zym). After 4 h, blood was drawn by cardiac puncture for circulating leukocyte counts (A). Peritoneal lavage was performed and leukocytes were counted (B), and lungs were harvested for myeloperoxidase (MPO) assay (C). Results are expressed as the mean ± SEM of at least five mice per group. ***p <0.001 versus saline-injected mice.

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Zymosan-induced peritonitis is predominantly due to neutrophil accumulation

To determine the types of leukocytes being recruited into the peritoneal cavity following zymosan challenge, we performed cytospins and leukocyte differential counts on the peritoneal exudate. Fig. 2 demonstrates that peritoneal leukocytosis was predominantly due to neutrophils (Fig. 2A). By contrast, the macrophage, lymphocyte and mast cell populations (Fig. 2B–D) were unaltered by zymosan, compared with saline-injected control animals.

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Figure 2. Types of leukocytes recovered in peritoneal exudate. Cytospins and leukocyte differentials were performed on the peritoneal lavage fluid obtained after i.p. challenge with saline or with 5 mg/kg zymosan (Zym) for 4 h. The percentages of neutrophils (A), macrophages (B), lymphocytes (C) and mast cells (D) were multiplied by the total cell number in the lavage fluid to obtain the number of peritoneal leukocyte subtypes. Results are expressed as the mean ± SEM of more than five mice per group. ***p <0.001 versus saline-injected mice.

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Kinetics of peritoneal leukocyte accumulation

To investigate the kinetics of leukocyte recruitment into the peritoneal cavity following zymosan challenge, we performed a time-course study. Mice were challenged with 5 mg/kg zymosan i.p. and, 0.5, 1, 2, 4 and 24 h later, circulating leukocyte counts, as well as leukocyte recruitment into the peritoneum, were quantified. As expected, treatment of wild-type mice with zymosan did not induce a significant change in circulating leukocyte counts across the various time points (Fig. 3A). There was, however, a progressive increase in leukocyte recruitment into the peritoneum over the first 4 h, which was maintained at 24 h (Fig. 3B). As shown in Fig. 3C, there was a tenfold increase in neutrophil counts as early as 2 h following challenge, reaching significance by 4 h. Neutrophil counts were reduced by ∼70% at 24 h, concomitant with a significant increase in peritoneal monocyte/macrophage infiltration (Fig. 3C and D).

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Figure 3. Kinetics of zymosan-induced leukocyte recruitment into the peritoneal cavity. C57BL/6 mice were challenged i.p. with saline (filled squares) or with 5 mg/kg zymosan (open squares). After 0.5, 1, 2, 4 or 24 h, blood was drawn by cardiac puncture for circulating leukocyte counts (A). Mice were sacrificed and peritoneal lavage was performed. Total leukocytes (B) as well as the number of neutrophils (C) and macrophages (D) in the exudate were quantified. Results are expressed as the mean ± SEM of at least five mice per group. **p <0.01, ***p <0.001 versus saline-injected mice.

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Zymosan-mediated peritonitis is TLR2 independent

Based on comprehensive in vitro studies, TLR2 has been identified as a dominant receptor for zymosan-induced inflammation 4, 5. As such, we were interested to determine whether the in vivo peritonitis response was TLR2 dependent. TLR2-deficient mice were treated with saline or challenged with 5 mg/kg zymosan i.p. for 4 or 24 h. As shown in Fig. 4A, leukocyte recruitment in response to zymosan was TLR2 independent. A similar trend was manifested in peritoneal neutrophil and monocyte accumulation, neither of which was significantly altered in zymosan-challenged TLR2–/– mice compared to wild-type controls (Fig. 4C and E). Although a ∼25% decrease in peritoneal leukocytes/neutrophils was noted, it did not reach significance, and certainly did not account for the majority of the zymosan response. The TLR2 independence of the zymosan response was not due to contaminating LPS, as TLR4-deficient mice had identical leukocyte recruitment profiles (Fig. 4B, D and F).

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Figure 4. Roles of TLR2 and TLR4 in neutrophil recruitment to the peritoneum. (A, C, E) C57BL/6 (filled bars) or TLR2–/– (unfilled bars); (B, D, F) C57BL/6 (filled bars) or TLR4-deficient C57BL/10ScNJ (unfilled bars) mice were challenged i.p. with saline or with 5 mg/kg zymosan (Zym) for 4 or 24 h. At the indicated times, mice were sacrificed, peritoneal lavage was performed and total leukocyte counts (A, B) as well as the number of neutrophils (C, D) and macrophages (E, F) in the exudate were quantified. Results are expressed as the mean ± SEM of at least five mice per group.

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Zymosan-mediated peritonitis is partially dependent upon LTB4

The leukotriene LTB4 is known to function as a potent neutrophil chemoattractant 22, and furthermore, LTB4 receptor-deficient mice have previously been shown to exhibit reduced peritoneal leukocyte accumulation in response to zymosan challenge 13. Thus, we were interested to determine whether pharmacological inhibition of LTB4 would similarly result in a significant inhibition of peritoneal leukocyte accumulation. To assess this, we pretreated C57BL/6 mice with the LTB4 antagonist CP-105,696 (750 µg/mouse i.p.) or with vehicle control 30 min prior to zymosan challenge. This concentration has previously been shown to inhibit neutrophil recruitment in a model of septic peritonitis 23. As shown in Fig. 5A and B, pretreatment with the LTB4 antagonist resulted in a significant reduction in both peritoneal leukocytosis and neutrophilia. Taken together, this suggests that LTB4 plays a role in zymosan-mediated peritonitis, but is not the dominant factor responsible for leukocyte recruitment.

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Figure 5. Effect of LTB4 inhibition on neutrophil recruitment to the peritoneum. C57BL/6 mice were pre-treated for 30 min with either vehicle control or with the LTB4 antagonist CP-105,696 (CP-105), and then challenged i.p. with 5 mg/kg zymosan (Zym). After 4 h, mice were sacrificed, peritoneal lavage was performed and total leukocyte counts (A), as well as the number of neutrophils (B) in the exudate were quantified. Results are expressed as the mean ± SEM of at least four mice per group, *p <0.05, **p <0.01, ***p <0.001 versus saline, #p<0.05 CP-105 versus vehicle.

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Early zymosan-mediated leukocyte recruitment is C3 dependent

Given that the in vivo peritonitis response to zymosan was independent of TLR2 and only partially LTB4 dependent, we were interested to determine which receptor was responsible for the majority of the observed effects. Zymosan is capable of activating complement 11, 24. To this end, we challenged C3-deficient mice with 5 mg/kg zymosan i.p. for 4 or 24 h. As shown in Fig. 6A, C3 is predominantly responsible for zymosan-induced peritoneal leukocyte recruitment, accounting for 75% of the infiltrate. Indeed, peritoneal leukocyte counts dropped from 18 × 106 in the C57BL/6 mice to 7 × 106 in the C3–/– mice following challenge with zymosan. Furthermore, as shown in Fig. 6B, C3-deficient mice showed significantly less neutrophil recruitment into the peritoneal cavity following 4 h zymosan challenge compared with wild-type mice, whereas at 24 h, monocyte recruitment was observed in the presence or absence of C3 (Fig. 6C).

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Figure 6. Effect of C3 deletion on neutrophil recruitment to the peritoneum. C57BL/6 (filled bars) or C3–/– (unfilled bars) mice were challenged i.p. with saline or with 5 mg/kg zymosan (Zym). After 4 or 24 h, mice were sacrificed, peritoneal lavage was performed and total leukocyte counts (A), as well as the number of neutrophils (B) and macrophages (C) in the exudate were quantified. Results are expressed as the mean ± SEM of at least five mice per group. ***p <0.001, C3–/–versus C57BL/6 mice.

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Complement-mediated peritoneal neutrophilia depends on C5aR

Given that the in vivo peritonitis response to zymosan was partially complement dependent, we determined which receptor was responsible for neutrophil accumulation. C5a is the dominant chemoattractant for neutrophils following complement activation 25. Thus, we challenged C5a receptor (C5aR)-deficient mice with 5 mg/kg zymosan i.p. for 4 or 24 h. As shown in Fig. 7A, C5aR is predominantly responsible for zymosan-induced peritoneal leukocyte recruitment at 4 h. Indeed, peritoneal leukocyte counts were significantly reduced in the C5aR–/– mice compared to wild-type controls following 4 h challenge with zymosan. Furthermore, as shown in Fig. 7B, C5aR-deficient mice had a ∼75% reduction in neutrophil recruitment into the peritoneal cavity following 4 h zymosan challenge compared with wild-type mice. In agreement with the findings observed in the C3-deficient mice, the 24 h response was C5aR independent.

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Figure 7. Effect of C5aR deletion on neutrophil recruitment to the peritoneum. C57BL/6 (filled bars) or C5aR–/– (unfilled bars) mice were challenged i.p. with saline or with 5 mg/kg zymosan (Zym). After 4 or 24 h, mice were sacrificed, peritoneal lavage was performed and total leukocyte counts (A), as well as the number of neutrophils (B) and macrophages (C) in the exudate were quantified. Results are expressed as the mean ± SEM of at least five mice per group. ***p <0.001, C5aR–/–versus C57BL/6 mice.

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Mast cell C5aR is required for zymosan-mediated neutrophil accumulation in the peritoneum

Mast cells have been implicated in zymosan-induced peritonitis 26, 27, and furthermore, they are known to express C5aR 28. Thus, we were interested to determine whether the mast cell C5aR was responsible for zymosan-mediated peritoneal neutrophilia. WBB6F1/J+/+ (control) mice had ∼2.4 × 105 peritoneal mast cells, whereas no mast cells were found in the peritoneal cavity of W/Wv mice (data not shown). Following zymosan challenge, there was a ∼50% reduction in the number of leukocytes/neutrophils recruited into the peritoneal cavity in the mast cell-deficient W/Wv mice compared with +/+ controls (Fig. 8A and B), in agreement with previously published reports 26, 27.

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Figure 8. The role of mast cells and mast cell C5aR in zymosan-mediated peritonitis. (A, B) WBB6F1/J+/+ control (filled bars) or W/Wv mast cell-deficient (unfilled bars) mice were challenged i.p. with saline or with 5 mg/kg zymosan (Zym). After 4 h, mice were sacrificed, peritoneal lavage was performed and total leukocyte counts (A), as well as the number of neutrophils (B) in the exudate were quantified. (C, D) W/Wv mice were reconstituted with either C57BL/6 or C5aR–/– BMMC. Four weeks later, mice were challenged with saline or with 5 mg/kg zymosan for 4 h, and peritoneal leukocytes (C) and peritoneal neutrophils (D) were quantified. Results are expressed as the mean ± SEM of at least five mice per group. **p <0.01, ***p <0.001 versus, ##p<0.01, ##p<0.001, W/Wv + C5aR–/– BMMC versus W/Wv + C57BL/6 BMMC.

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To examine whether mast cell C5aR was responsible for zymosan-induced leukocyte recruitment into the peritoneum at 4 h, we cultured bone marrow-derived mast cells (BMMC) from C57BL/6 or C5aR–/– mice and used these cells to reconstitute mast cell-deficient (W/Wv) mice. Four weeks after BMMC transfer, mice were challenged with 5 mg/kg zymosan (i.p. × 4 h). As shown in Fig. 8C and D, zymosan-challenged mast cell-deficient mice reconstituted with C5aR–/– mast cells had significantly reduced levels of leukocyte/neutrophil recruitment compared to mast cell-deficient mice reconstituted with wild-type mast cells, or mast cell sufficient (WBB6F1/J+/+) mice. Similar numbers of mast cells were found in mice reconstituted with wild-type (3.8 ± 0.5 × 105) or C5aR–/– (2.4 ± 0.5 × 105) mast cells. Taken together, this suggests that zymosan-induced mast cell activation is mediated by C5aR.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

We have demonstrated robust neutrophil and monocyte recruitment into the peritoneal cavity 4 and 24 h, respectively, following systemic administration of zymosan. Interestingly, in our system, TLR2 was not responsible for the in vivo leukocyte recruitment in response to zymosan. In fact, early neutrophil accumulation was largely dependent upon complement. This was demonstrated in mice lacking complement factor 3, a protein central to the activation of the classical, alternative and lectin pathways. Moreover, the receptor for C5a, a potent chemoattractant for neutrophils, played a dominant role at 4 h, and in its absence, zymosan-induced peritoneal neutrophilia was reduced by more than 70%. Interestingly, the levels of residual peritoneal neutrophil recruitment observed in the C3- and C5aR-deficient mice were similar, suggesting that C5a is the dominant complement product/effector generated following zymosan challenge in vivo. In agreement with the work of Rao et al.12 and Haribabu et al.13, early peritoneal leukocyte recruitment was also partially dependent upon LTB4, as mice pretreated with an LTB4 antagonist prior to zymosan challenge exhibited a significant reduction in peritoneal leukocytosis. By 24 h, a second phase of leukocyte recruitment occurred, which was predominantly monocytic in nature. Surprisingly, peritoneal monocyte accumulation following 24 h zymosan challenge was completely independent of TLR2 or complement. Interestingly, the early neutrophil recruitment was not responsible for the later monocytic recruitment, suggesting that monocytes are not dependent upon neutrophil-derived molecules. In agreement with our finding, Getting et al.29 have previously shown that pharmacological inhibition of zymosan-induced peritoneal neutrophilia did not abrogate subsequent monocyte recruitment.

TLR2 has been identified as the dominant receptor for in vitro zymosan responsiveness 4, 5. However, those studies primarily utilized macrophage cell lines. Of particular interest, although the macrophage is the preferred cell type for the in vitro assessment of zymosan-mediated TLR2 activation, peritoneal leukocytosis is enhanced following zymosan challenge in macrophage-deficient mice 27. Furthermore, we have demonstrated that neutrophil accumulation in zymosan-induced peritonitis was TLR2 independent. In direct contrast to our work, a dominant role for TLR2 has been found in zymosan-induced arthritis and pleurisy 6, 7. We hypothesize that this discrepancy is due to the use of different model systems rather than zymosan source (Sigma) or dosage, which was similar in our study (5 mg/kg zymosan, which is equivalent to 125-150 μg/mouse), and those of others 6, 7, 27. Frasnelli et al.7 showed a dominant role for TLR2 and no role for complement in zymosan-induced arthritis. It is important to note that zymosan-induced arthritis is a very different model of inflammation than peritonitis, and has a significant adaptive immune pathway component. Furthermore, as the zymosan is being administered into a joint cavity, this model could (similar to in vitro work) allow TLR2 to dominate, as complement levels in this site are much lower than in plasma 30. In the case of Takeshita et al.6, this study used a 4 h model of pleurisy and found that neutrophil accumulation following zymosan challenge could be ablated by ∼80% when mice were pretreated with a blocking antibody against TLR2 or were deficient in the downstream adaptor protein, MyD88. Moreover, recruitment into the pleural cavity was dependent upon mast cells, and mast cell-expressed MyD88. In addition, Takeshita et al. demonstrated that pleural neutrophil recruitment was dependent upon activation of the H4 histamine receptor, whereas we observed predominantly C5aR-dependent peritoneal leukocyte recruitment, although both receptors are expressed by mast cells. It is possible, that mast cell-expressed H4 and C5a receptors may function cooperatively in certain models of zymosan-mediated leukocyte recruitment. However, taken together, our data along with those of Takeshita et al. also highlight the potential differences in neutrophil recruitment in different organs.

The complement system is important in host defense and clearance of infection (reviewed in 31), and is known to be activated in a cell-independent manner by zymosan 20. Thus, we hypothesized that complement would mediate the TLR2-independent peritoneal leukocytosis following zymosan administration. Interestingly, although complement played a dominant role, loss of either C3 or C5aR did not completely ablate early peritoneal neutrophilia, and had no effect on peritoneal monocyte accumulation 24 h following zymosan challenge. Thus, we would propose that additional mechanisms exist to facilitate peritoneal leukocytosis in the absence of complement. One possibility is that the minor reduction (25%) seen in TLR2-deficient mice could account for the small remaining leukocyte recruitment observed in C3–/– and C5aR–/– mice. Indeed, essentially all in vitro work has been done in the absence of plasma and complement, to unmask a role for TLR2. Another possible candidate receptor is dectin-1. Brown et al.14 have shown that, although CR3 is important in recognition of opsonized zymosan, dectin-1 functions as a dominant receptor for unopsonized zymosan. Interestingly, many of the pro-inflammatory functions of dectin-1 are collaborative with TLR2 15. As we did not see a dominant role for TLR2 in our model, either dectin-1 (the purported partner for TLR2) also did not contribute to the peritoneal leukocytosis or, alternatively, dectin-1 can work independently of TLR2. A final possibility is that an as yet, unidentified pathway is involved.

Lastly, we have shown a role for complement-mediated mast cell activation in zymosan-induced peritoneal neutrophil accumulation (at 4 h). Mast cells have been reported to express C5aR and are known to be activated by complement 28, 32. Indeed, using mast cell reconstitution of W/Wv mice, we found that mast cell C5aR was important for zymosan-induced peritoneal neutrophilia. It is worth noting, however, that the reconstitution experiments did not inhibit peritoneal neutrophilia to the same extent as complete C5aR deficiency, suggesting that other cell types may be activated via C5aR to promote neutrophil influx. Indeed, C5aR is expressed by neutrophils 33 and, therefore, these cells could be directly recruited into the peritoneum following zymosan challenge and accompanying C5a production. In addition, endothelium can be directly activated by C5a, resulting in P-selectin up-regulation 34, as well as the production of chemokines, including KC and MIP-2 28, which could further facilitate leukocyte recruitment. In fact, endothelium would need to be activated either by the mast cells or by zymosan to initiate neutrophil recruitment, as in the absence of endothelial adhesion molecules, zymosan-mediated neutrophil recruitment is significantly impaired 24.

Thus, we conclude from this work that the in vitro paradigm of TLR2 dominance for zymosan responsiveness is not unequivocally true in all in vivo scenarios. We would acknowledge that the majority, if not all, in vitro studies were done in the absence of plasma and complement to unmask the role for TLR2. Moreover, we do not dispute that TLR2 is important in certain settings; however, we suggest that it is not essential for the early neutrophil mobilization into the peritoneum following challenge with zymosan. Indeed, we propose that, in a model of peritonitis, zymosan challenge results in TLR2-independent activation of the complement pathway resulting in the generation of the anaphylatoxin C5a, which then interacts with its cognate receptor on the surface of multiple cell types including, but not limited to, the mast cell. C5aR activation then results in neutrophil recruitment from the circulation and into the peritoneal cavity. The leukocyte recruitment response would be further potentiated via LTB4 produced by both peritoneal resident cells and by recruited neutrophils.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

C57BL/6, TLR2-deficient, TLR4-deficient (C57BL/10ScNJ), C3-deficient (B6.129S4-C3tm1Crr/J) and mast cell-deficient WBB6F1/J-KitW/KitW–v (W/Wv) plus control (WBB6F1/J+/+) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C5aR-deficient mice were a kind gift of Dr. Craig Gerard (Harvard Medical School, Boston, MA). Mice were on a C57BL/6 background, unless otherwise noted. Mice were used at more than 6 weeks of age, and were housed in a pathogen-free facility. Animal protocols were approved by the University of Calgary Animal Care Committee and were in accordance with the Canadian Guidelines for Animal Research.

Reagents

Zymosan was purchased from Sigma (Oakville, Ontario, Canada) as in 6, 7. The LTB4 antagonist CP-105,696 was a gift from Pfizer (Groton, CT). RPMI 1640, glutamine, sodium pyruvate and penicillin/streptomycin were from Invitrogen (Burlington, Ontario, Canada). Heat-inactivated FBS was purchased from Hyclone (Logan, UT). Recombinant murine IL-3 and SCF were purchased from R&D Systems (Minneapolis, MN).

Circulating leukocyte counts and experimental peritonitis

Zymosan was prepared in sterile PBS, stored at –80°C and injected i.p. in a final volume of 250 μL following 1 h sonication. At the noted times post-injection, mice were anesthetized with Isoflurane (Bimeda-MTC, Cambridge, Ontario, Canada) and whole blood was collected via cardiac puncture for circulating leukocyte counts. Mice were sacrificed, and a peritoneal lavage was performed using 3 mL of PBS. Exudate was recovered following a 60-s gentle manual massage. Cell counts were determined with a hemacytometer, and lavage fluid was cytospun and stained with Wright-Giemsa. Leukocyte differentials were determined from a count of 200 cells. The percentage of cells was then multiplied by the total cell number in the lavage fluid to obtain the number of peritoneal leukocyte subsets.

Lung myeloperoxidase assay

Myeloperoxidase (MPO) activity was used as a measure of neutrophil recruitment into the pulmonary microvasculature 21, 35. Lungs were harvested at the end of each experiment and frozen at –80°C. Samples were processed using hexadecyl-trimethylammonium bromide and dianisidine-H2O2. The change in absorbance at 450 nm in the 96-well plates was determined for 60 s using a kinetic microplate reader (Molecular Devices Corporation, Sunnyvale, CA).

Bone marrow-derived mast cells (BMMC)

Bone marrow was isolated from the femurs and tibias of 6–8-week-old male C57BL/6 and C5aR-deficient mice. Red blood cells were lysed with ACK buffer, and the remaining cells were washed and maintained in RPMI 1640 medium with 10% FBS, sodium pyruvate, glutamine, penicillin/streptomycin, 2-mercaptoethanol, 10 ng/mL IL-3 and 12.5 ng/mL SCF. Cells were transferred into new flasks on a weekly basis to remove any adherent cells. BMMC were transferred into recipient mice after a minimum of 6 weeks in culture (at ∼98% purity as determined by flow cytometry and Wright-Giemsa staining). For reconstitution, BMMC (5 × 106 in 250 µL sterile saline) were injected i.p. into W/Wv mice. Age-matched wild-type littermate control mice were injected with 250 μL sterile saline. Mice were housed for an additional 4 weeks before being challenged with saline or zymosan.

Statistics

Results are expressed as mean ± SEM. A one-way ANOVA and a t-test with Bonferroni correction were used for multiple comparisons. Statistical significance was set at p <0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This work was funded by the Canadian Institutes of Health Research (CIHR) and a CIHR group grant. P. Kubes is a Canadian Research Chair recipient, the Joan Snyder Chair in Critical Care Medicine and an Alberta Heritage Foundation for Medical Research Scientist. S.C. Mullaly is funded by the Heart and Stroke Foundation of Canada and a Province of Alberta Graduate Scholarship.

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