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Blood, 1 June 2007, Vol. 109, No. 11, pp. 4846-4855. Prepublished online as a Blood First Edition Paper on February 8, 2007; DOI 10.1182/blood-2006-09-045641.
IMMUNOBIOLOGY Involvement of mast cells in IL-12/23 p40 production is essential for survival from polymicrobial infections1 Atopy (Allergy) Research Center 2 Department of Immunology, and 3 Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; 4 Biotechnology Research Center, University of Tokyo, Japan
Interleukin-12 (IL-12), a heterodimeric cytokine (p35/p40) produced mainly from macrophages and dendritic cells, is an important regulator of T-helper 1 cell responses and for host defense. We found that interferon (IFN) consensus sequence binding protein (ICSBP), which is a transcription factor essential for the expression of p40, was expressed in mouse bone marrow–derived mast cells (BMMCs). The transcription levels of p35 and p40 were increased by stimulation of BMMCs with IFN-  /lipopolysaccharide (LPS). IL-12 was secreted from BMMCs in response to LPS but not by Fc RI cross-linking. The p40 levels in the peritoneal cavity of mast cell–deficient W/Wv and W/Wv reconstituted with p40–/– BMMCs were significantly lower than those of WBB6F1+/+ and wild-type (WT) BMMC-reconstituted W/Wv in the acute septic peritonitis model. The survival rate of W/Wv reconstituted with p40–/– BMMCs was significantly decreased compared to those of WBB6F1+/+ and WT-BMMC–reconstituted W/Wv, which was due to reduced production of IFN- and subsequent impaired activation of neutrophils in the peritoneal cavity. Survival rate of p40–/– mice was also restored by adoptive transfer of WT-BMMCs. These results demonstrate that mast cells play a significant role in the production of IL-12 required for host defense. This is the first report to demonstrate that mast cells are a crucial source of functional IL-12.
Interleukin-12 (IL-12) is a cytokine that governs production of interferon-gamma (IFN- ) in natural killer (NK) cells and CD4+ T cells1,2 and that is involved in the induction and maintenance of T-helper 1 (Th1) cells.3–5 IL-12 also plays important roles in resistance against various infectious agents including viruses, bacteria, and parasites.6,7 The deficiency of IL-12 in knock-out mice causes a severely depressed Th1 response, supporting the role of IL-12 in the Th1 response and resistance to infections.3,8,9 IL-12 (p70) is a heterodimer composed of 2 subunits, p40 and p35, which are encoded by 2 separate genes.10 In many cell types, p35 expression is induced by stimulation with pathogens or their components, such as Gram-negative bacterial lipopolysaccharide (LPS).11 p40 expression is also induced by stimulation of LPS, but is limited to macrophages, dendritic cells, B cells, and neutrophils, among which the first 2 cells are the primary sources of IL-12.1,12–14 The p40 protein also forms a heterodimer with a p35-related protein p19.15 The heterodimer p19/p40, known as IL-23, a member cytokine of the IL-12 family, also regulates Th1-cell responses.16–18 Thus, the p40 protein is considered to be an IL-12–specific subunit required for the functional expression of IL-12.
IFN consensus sequence binding protein (ICSBP), also designated IFN regulatory factor 8 (IRF-8), is a nuclear transcription factor belonging to the IRF family. ICSBP–/– mice cannot show Th1-mediated responses because of a deficiency in IL-12 production.19–21 Several previous studies indicate that ICSBP binds to the Ets site of the p40 promoter to up-regulate promoter activity in response to IFN- We have analyzed the regulation mechanisms of hematopoietic cell development by transcription factors and found (1) cooperation between transcription factors PU.1 and GATA-1, and (2) repression of GATA-1–dependent transactivation by cofactor FOG-1, which causes mast cell–specific gene expression.25–28 In addition, the expression level of PU.1 determines the fate of cell differentiation between mast cells and monocytes.29–31 Although ICSBP is one of the partners of PU.1, the regulatory mechanism for the expression of ICSBP in mast cells and the involvement of ICSBP in the cell fate determination between mast cells and monocytes are unclear. Mast cells play roles not only as effector cells in allergic disease but also as initiator cells of innate and acquired immune responses against various pathogens.32–35 The innate immune response is initiated by Toll-like receptors (TLRs). Recent reports have demonstrated that mast cells express TLRs and associate with innate immune responses against Gram-negative bacteria via TLR4, a signal transducer of LPS.36,37 In other reports, the mRNAs for the IL-12 components were detected in mouse bone marrow–derived mast cells (BMMCs)38 and in LPS-stimulated human mast cells.39 These observations suggest that mast cells produce IL-12 to contribute to innate immunity. However, IL-12 production in mast cells has not been well analyzed, and the relationship between IL-12 production in mast cells and innate immunity is unknown.
Here, we found that ICSBP is expressed in BMMCs in response to LPS, similar to macrophages and dendritic cells. We also demonstrated that IL-12 is inducibly produced from BMMCs upon LPS stimulation, and that this production increases in the presence of IFN-
Mice BALB/c, C57BL/6, WBB6F1+/+, and mast cell–deficient WBB6F1-W/Wv mice were purchased from Japan SLC (Hamamatsu, Japan). IL-12/23 p40-deficient (p40–/–) mice (B6.129S1-IL12btm1Jm/J) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were performed according to the approved manual of the institutional review board of Juntendo University, Japan. Preparation of BMMCs
BMMCs were generated from the femoral bone marrow cells of female mice as described previously.40 Cells were incubated in RPMI 1640 (Sigma-Aldrich, St Louis, MO) supplemented with 10% heat-inactivated FCS (Biological Industries, Haemek, Israel), 100 U/mL penicillin, 100 µg/mL streptomycin, 100 µM 2-mercaptoethanol, 10 mM sodium pyruvate, 10 µM MEM nonessential amino acid solution (Invitrogen, Carlsbad, CA), 100 U/mL murine IL-3 (PeproTech EC, London, United Kingdom), and 0.5 U/mL murine stem cell factor (SCF; PeproTech EC) at 37°C in a humidified atmosphere in the presence of 5% CO2. After 4 to 5 weeks of culture, more than 96% of the cells were identifiable as mast cells as determined by toluidine blue staining and fluorescence-activated cell sorting (FACS) analysis of cell surface expression of c-Kit and Fc FACS sorting of BMMCs
The 4-week–cultured BMMCs were stained with FITC-conjugated anti–mouse Fc Peritoneal macrophages and macrophage cell line
Murine peritoneal macrophages (PEC-M Reverse-transcription–polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from either BMMCs or RAW264.7 cells using STAT-60 (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. First-strand cDNA was constructed from 1 µg total RNA with oligo(dT)12-18 as a primer using Superscript II RNase H– reverse transcriptase (Life Technologies, Rockville, MD). PCR was performed using specific primer sets for mouse ICSBP (GenBank accession no. NM_008320: 5'-GCG TGG GAA CCG GCG GCA GGA TG and 5'-CTC AGG CGA GGT GGG GTG CCC GGA C),41 mouse p40 (accession no. M86671: 5'-ATG TGT CCT CAG AAG CTA ACC and 5'-CTA GGA TCG GAC CCT GCA GGG AAC), mouse p35 (accession no. M86672: 5'-ATG TGT CAA TCA CGC TAC CTC C and 5'-TCA GGC GGA GCT CAG ATA GCC),42 mouse p19 (accession no. AF301619: 5'-ATG CTG GAT TGC AGA GCA GTA ATA ATG C and 3'-TTA AGC TGT TGG CAC TAA GGG CTC),15 and species nonspecific GAPDH (5'-AGT ATG ACT CCA CTC ACG GCA A and 5'-TCT CGC TCC TGG AAG ATG GT).43 Quantitative PCR Expression level of each mRNA was quantitatively analyzed by a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) with cDNA prepared as described above under "RT-PCR analysis" and TaqMan Universal Master Mix (Applied Biosystems). The expression level of ICSBP mRNA detected by TaqMan Gene Expression Assays (Mm00492567_m1) was shown as the ratio to that of GAPDH, which was determined with Rodent GAPDH Control Reagents (Applied Biosystems), by calculation of cycle threshold (Ct) values in amplification plots with a 7500 SDS software (Applied Biosystems). Western blot analysis of ICSBP A total of 2.5 x 106 cells of BMMCs or RAW264.7 was lysed by addition of 1 x sampling buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 0.1 mg/mL bromphenol blue dye, and 10% 2-mercaptoethanol). The cell lysates were electrophoretically resolved in 10% SDS–polyacrylamide gel and then transferred onto the PVDF membrane (Millipore, Bedford, MA). ICSBP and YY1 were probed by anti-ICSBP goat polyclonal antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-YY1 mouse monoclonal antibody (H-10; Santa Cruz Biotechnology), respectively. Alexa Fluor 680–conjugated anti–goat IgG or anti–mouse IgG (Molecular Probes, Eugene, OR) was used as the secondary antibody. Infrared fluorescence on the membrane was detected by an infrared imaging system Odyssey (LI-COR Biosciences, Lincoln, NE). Measurement of cytokine concentration
BMMCs or PEC-M Mast cell reconstitution in W/Wv mice Mast cell deficiency of 5-week-old W/Wv mice was reconstituted by intraperitoneal injection of 2 x 106 BMMCs from C57BL/6 or p40–/– mice as previously described.33,37,40 The mice were used for experiments at 5 weeks after injection of BMMCs. Reconstitution of mast cells was confirmed by toluidine blue or Alcian blue and safranin staining of the cytospun preparation of peritoneal cells. Blood was collected from the tail into EDTA-coated microcapillary tubes (20-30 µL/sampling) and red cell counts were determined manually. The hematocrit level was determined after centrifugation of blood at 15 000g for 3 minutes. Cecal ligation and puncture Cecal ligation and puncture (CLP) was performed as previously described.37,44,45 In brief, mice were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital (Abbott Laboratories, Abbott Park, IL). The cecum was exposed by a 1-cm midline incision on the anterior abdomen and subjected to ligation of the distal half followed by a single puncture with a 0.9-mm needle. The cecum was then squeezed gently, replaced into the abdomen, and closed using a nylon suture for operation. Mice were observed for mortality every day over a period of 14 days. Estimation of cytokine concentration in peritoneal fluids and serum Peritoneal exudates were collected from CLP-induced mice at the indicated time points. The concentrations of cytokines in peritoneal fluids were determined by ELISA kits according to the manufacturer's instructions (BD PharMingen). Bacterial counts in the peritoneal exudates The number of bacteria in the peritoneal cavity was determined by previously described method.46 In brief, peritoneal fluids collected from mice, which were killed 6 hours after sepsis induction, were placed onto Mueller-Hinton agar dishes (Difco, Hunt Valley, MD). After 18-hour incubation at 37°C, colony-forming units (CFUs) were scored. The results were expressed as log10 of CFUs/mL. Count of neutrophil migrated to the peritoneal cavity Peritoneal exudates were collected from CLP-treated mice at 0 or 6 hours after sepsis induction, and total cell numbers were counted. Differential cell counts were conducted on cytospun preparations prepared from each fluid with the May-Grünwald-Giemsa staining (Muto Pure Chemicals, Tokyo, Japan). The results were expressed as the number of neutrophils per cavity. IL-12 and IL-23 treatment W/Wv mice were injected intraperitoneally with 25 ng recombinant murine IL-12 or IL-23 (R&D Systems, Minneapolis, MN) every 5 days after CLP treatment according to the method reported previously.47,48 Statistical analysis Statistical analysis of most data was performed using Student t test, and data are presented as mean ± SD. Statistical analysis of survival data in CLP experiments was performed using the log-rank test.
ICSBP expression in BMMCs
ICSBP, the expression of which is transactivated by IFN-
Transcription of IL-12–related molecules in BMMCs
The p40 promoter is known to be transactivated by ICSBP, and the recruitment of ICSBP to a critical Ets site is enhanced by IFN-
IL-12 secretion from BMMCs upon LPS stimulation
Transcription of both p40 and p35 was observed in LPS-stimulated BMMCs. Then, to examine the IL-12 secretion from mast cells, we measured the levels of p40 and p70 in culture supernatant by ELISA kits (Figure 3). As shown in Figure 3A, BMMCs secreted p40 by LPS stimulation in a dose-dependent manner, whereas unstimulated BMMCs had a p40 secretion level below the detection limit. The secretion levels from BMMCs upon LPS stimulation were greatly enhanced by IFN-
Effect of IFN- released from BMMCs on IL-12 and ICSBP production
As described in Figure 3, BMMCs secreted p40 and p70 by LPS stimulation even in the absence of IFN-
Mast cells are a source of p40 in a model of acute septic peritonitis We had confirmed that mast cells produce IL-12 by LPS stimulation in vitro. To evaluate the involvement of mast cells as a source of IL-12 in vivo, we analyzed the cytokine production levels in peritoneal fluids of mice with CLP, a mouse model of acute septic peritonitis. Peritoneal mast cells of mast cell–deficient W/Wv mice were reconstituted with BMMCs either from wild-type (WT) C57BL/6 or p40–/– mice. The reconstitution of peritoneal mast cells derived from W/Wv mice that received BMMCs from WT mice and p40–/– mice was confirmed by positive staining with both Alcian blue and safranin, a feature of connective tissue–type mast cells (data not shown). In addition, we determined hematocrit value and red cell count of each mouse to analyze whether non–mast cell hematopoietic reconstitution initiated by pluripotential c-kit+ progenitors, which may be contaminated in BMMC preparations, occurred in this reconstitution model. W/Wv mice were confirmed to be anemic, with low hematocrit value and red cell count compared with WBB6F1+/+ mice (Table 1). Reconstitutions with WT-BMMCs and with p40–/– BMMCs had no positive effect on hematocrit value and red cell count (Table 1), suggesting that protective effects by non–mast cell hematopoietic reconstitution were not apparently observed in this experiment.
As shown in Figure 5A, a 35% reduction in p40 levels in peritoneal fluids was observed for unreconstituted W/Wv mice compared with congeneric normal WBB6F1+/+ mice, by 5-hour incubation after CLP induction. The reduction in p40 levels of W/Wv mice was not recovered by reconstitution with p40–/– BMMCs, but recovered by reconstitution with WT-BMMCs. In the serum, the p40 levels were reduced by 39% for unreconstituted W/Wv and W/Wv mice reconstituted with p40–/– BMMCs when compared with WBB6F1+/+ and W/Wv mice reconstituted with WT-BMMCs (data not shown). These observations indicate that mast cells are a source of p40 in this model. Previous studies demonstrated that an increase in peritoneal TNF-  level is critical for neutrophil recruitment and bacterial clearance following CLP and that mast cells are an important source of peritoneal TNF-.33,44,53 IL-6 produced by mast cells is also an important cytokine in local inflammatory reactions by amplifying leukocyte recruitment.37,54 Therefore, we determined the TNF- and IL-6 levels by CLP induction. By 5 hours of incubation after CLP induction, the levels of TNF- (Figure 5B) and IL-6 (Figure 5C) in the peritoneal fluids of W/Wv mice reconstituted with p40–/– BMMCs were the same as those of WBB6F1+/+ and W/Wv mice reconstituted with WT-BMMCs, whereas that of unreconstituted W/Wv was markedly lower (50% or less of WBB6F1+/+ and W/Wv mice reconstituted with WT-BMMCs). These observations indicate that the reductions in TNF- and IL-6 levels in the peritoneal cavity of W/Wv mice by CLP were recovered by reconstitution both with WT-BMMCs and p40–/– BMMCs, suggesting no role of IL-12 in TNF- and IL-6 production.
W/Wv mice reconstituted with p40–/– BMMCs exhibit decreased survival compared with WT-BMMC–reconstituted mice IL-12 plays multiple important roles in bacterial clearance and survival in CLP-mediated polymicrobial infections.55–57 We examined the effects of p40 deficiency in mast cells on survival after CLP. As shown in Figure 6, in CLP-induced acute septic peritonitis, the W/Wv mice reconstituted with p40–/– BMMCs showed a significantly higher mortality rate than the WBB6F1+/+ mice and the W/Wv mice reconstituted with WT-BMMCs, whereas the W/Wv mice without reconstitution with BMMCs showed the highest mortality rate. At 14 days after CLP, 80% of the W/Wv mice reconstituted with p40–/– BMMCs had died, whereas the WBB6F1+/+ mice and the W/Wv mice that carried mast cells expressing IL-12 showed an approximately 80% survival rate. This indicates that IL-12 produced by mast cells contributes to host survival in this model.
Effects of IL-12 produced from mast cells on bacterial clearance–related factors
To elucidate the role of IL-12 produced from mast cells in host survival, we analyzed the bacterial counts in the peritoneal cavity of mice after CLP surgery. The CFU in peritoneal exudates of W/Wv mice was significantly higher than that of WBB6F1+/+ mice (Figure 7A). The reduced bacterial clearance of W/Wv mice was normalized by the reconstitution with WT-BMMCs, whereas the reconstitution with p40–/– BMMCs partially restored the reduced bacterial clearance, which was observed in W/Wv (Figure 7A). We then investigated the neutrophil recruitment to the peritoneal cavity, because neutrophils rapidly migrate toward the infection site and play important roles in host defense by releasing a variety of microbicidal mediators. W/Wv mice showed an impaired neutrophil migration compared with WBB6F1+/+ mice (Figure 7B). The neutrophil migration was restored to the level comparable to that of WT mice by WT-BMMC or p40–/– BMMC reconstitution (Figure 7B). This result coincides with the above data that the concentration of TNF-
Effect of adoptive transfer of WT-BMMCs on survival of CLP-treated p40–/– mice The p40-deficient mice have defects in clearing polymicrobial infections in CLP experiments.46 To evaluate mast cells as a source of IL-12, we compared the survival rates of p40–/– mice with and without adoptive transfer of WT-BMMCs in the CLP experiment. The survival rate of p40–/– mice exposed to CLP was 0% at 2 days after surgery (Figure 8A). In contrast, p40–/– mice transferred with WT-BMMCs by intraperitoneal injection in the same way as reconstitution of W/Wv showed 50% survival rate at 14 days after surgery (Figure 8A). This result suggests that mast cells function as a source of p40, which partially recovers the p40 deficiency of the knock-out mice.
Effects of IL-12 and IL-23 on survival of CLP-treated W/Wv mice The p40 protein is also a constituent of IL-23 when forming a complex with a p35-related protein, p19. Because involvement of IL-23 in host defense against infectious diseases is also reported,18,58 we examined the effects of recombinant murine IL-12 and IL-23 on survival rates of CLP-treated W/Wv mice. When W/Wv mice were injected intraperitoneally with IL-12 at every 5 days after CLP surgery, 50% of mice were alive after 14 days, whereas all W/Wv mice without cytokine treatment were dead at 2 days after surgery (Figure 8B). When W/Wv mice were injected intraperitoneally with IL-23, the survival rate was the same as that of mice that had been injected with IL-12 cytokine for 5 days; the rate fell to 20% after 14 days (Figure 8B). This result suggests that defect of host survival due to the absence of mast cells is partially rescued by infection of recombinant IL-12 and IL-23.
In this study, we demonstrated that mouse BMMCs produce IL-12–related molecules, in response to LPS, and that this production is augmented by preincubation of the cells with IFN- . Furthermore, in an acute peritonitis model, we showed that mast cells serve as a source of IL-12 to contribute to bacterial clearance. These observations indicate a novel role for mast cells in innate immune responses to bacterial infections.
In the transcription of p40 induced through TLR4-mediated stimulation by LPS, ICSBP is an essential transcription regulatory factor in macrophages, B lymphocytes, and activated T lymphocytes, and the expression of ICSBP is increased by IFN-
Mast cells play a prominent role in the early immune response to invading pathogenic bacteria.32–35 The mast cells secrete various mediators including TNF-
Reduced survival rate of p40 knock-out mice is partially rescued by adoptive transfer of WT-BMMCs (Figure 8A), also suggesting that mast cells are one of the important sources of p40. However, since the survival rate is not completely normalized by the transfer, the role of mast cells as a source of IL-12 may be distinguished from those of macrophages and dendritic cells. Alternatively, the transferred mast cells could not fully recover the production of enough IL-12 in the peritoneal cavity of IL-12–deficient mice. In addition, treatment of IL-12 partially rescued the reduced survival rate of mast cell–deficient mice (Figure 8B). This observation indicates that deficiency of mast cells is not fully complemented by presence of IL-12, and that TNF- In infectious diseases, IL-23 also plays an important role in host defense, although this role(s) has not been fully clarified.18,58 We were unable to define the presence and/or the amount of IL-23 in the culture media of mast cells because of the absence of an IL-23 ELISA system, and, therefore do not rule out the involvement of IL-23 produced by mast cells in the resistance to CLP-induced acute septic peritonitis in this investigation. In CLP experiment, IL-23 treatment partially rescues survival rate of W/Wv mice (Figure 8B), suggesting that IL-23 may have a protective effect on the bacterial clearance reduced by mast cell deficiency. Further study is required to elucidate the involvement of IL-23. In any case, this study demonstrates that mast cells as well as dendritic cells and macrophages are an important source of p40 during polymicrobial infections, and that they contribute to bacterial clearance through the production of p40. Although mast cells had been characterized as a source of Th2 cytokines such as IL-4, IL-5, and IL-13, our results indicate that mast cells have bilateral characteristics that are able to give the signal to induce the production of either Th1 or Th2 cytokines depending on the environment of the cells. This hypothesis is not only important for understanding innate immune responses to bacterial infections, but is also useful in clarifying the roles of mast cells in other skin diseases caused by Th1 dominancy, including psoriasis and contact sensitivity, in which p40 plays a pivotal role in the pathogenesis,64,65 and in the infiltration of mast cells in skin lesions.66
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Abstract
Introduction
s) were obtained from peritoneal exudate cells of female BALB/c mice as described previously.
s at a concentration of 2 x 105 cells in 150 µL complete medium were incubated in the presence or absence of 100 U/mL murine IFN-
), W/Wv mice (10 mice/group; 
), and W/Wv mice reconstituted with wild-type BMMCs (8 mice/group; 
) or p40–/– BMMCs (9 mice/group; 
) following CLP induction are shown. Statistical analysis was performed using the log rank test. *P < .05, significantly different from the WBB6 F1+/+ mice group. These results are representative of 3 independent experiments that had similar results.
), wild-type BMMC–reconstituted WBB6 F1-W/Wv (
), and p40–/– BMMC–reconstituted WBB6 F1-W/Wv mice (
), wild-type BMMC–reconstituted WBB6 F1-W/Wv (hatched bar), and p40–/– BMMC–reconstituted WBB6 F1-W/Wv mice (dotted bar) in panels B-C. Data represent mean ± SD of 5 mice and *P < .05; significantly different from the mean value of the corresponding control in panels B-C. (C) Concentration of IFN-
B activation, which is mediated by IFN-