International Journal of Molecular Sciences Article IL-10 Modulates the Expression and Activation of Pattern Recognition Receptors in Mast Cells Roberto Riquelme-Neira 1,2 , Romina Walker-Vergara 1 , Joan Antoni Fernández-Blanco 2 and Patrocinio Vergara 2, * 1 Núcleo de Investigaciones Aplicadas en Ciencias Veterinarias y Agronómicas, Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, Sede Concepción, Chacabuco 539, Concepción 4070254, Chile; rriquelmen@udla.cl (R.R.-N.); romina.walker@edu.udla.cl (R.W.-V.) 2 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; joanantoni.fernandez@gmail.com * Correspondence: patri.vergara@uab.es Abstract: Mast cells (MCs) are involved in several immune-related responses, including those in bacterial infections, autoimmune diseases, inflammatory bowel diseases, and cancer, among others. MCs identify microorganisms by pattern recognition receptors (PRRs), activating a secretory response. Interleukin (IL)-10 has been described as an important modulator of MC responses; however, its role in PRR-mediated activation of MC is not fully understood. We analyzed the activation of TLR2, TLR4, TLR7 and Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mucosal-like MCs (MLMCs) and peritoneum-derived cultured MCs (PCMCs) from IL-10−/− and wild-type (WT) mice. IL-10−/− mice showed a reduced expression of TLR4 and NOD2 at week 6 and TLR7 at week 20 in MLMC. In MLMC and PCMC, TLR2 activation induced a reduced secretion of IL-6 and TNFα in IL-10−/− MCs. TLR4- and TLR7-mediated secretion of IL-6 and TNFα was not detected in PCMCs. Finally, no cytokine release was induced by NOD2 ligand, and responses to TLR2 and TLR4 were lower in MCs at 20 weeks. These findings indicate that PRR activation in MCs depends on the phenotype, ligand, age, and IL-10. Keywords: interleukin-10; MMC; PCMC; PRR; NOD2; TLR Citation: Riquelme-Neira, R.; Walker-Vergara, R.; Fernández-Blanco, J.A.; Vergara, P. IL-10 Modulates the Expression and Activation of Pattern Recognition 1. Introduction Receptors in Mast Cells. Int. J. Mol. Mast cells (MCs) are long-lived, tissue-localized immune cells that originate from Sci. 2023, 24, 9875. https://doi.org/ pluripotent progenitors in the bone marrow [1–3]. Immature mast cells migrate to periph- 10.3390/ijms24129875 eral tissues to complete their maturation and differentiation, mainly in those tissues that Academic Editor: Davide Firinu are in proximity to host-environment interfaces, such as the skin, gastrointestinal tract, and airways, having different functions and phenotypes depending on their location [4–6]. Received: 25 April 2023 Accordingly, they are classified as mucosal MCs (MMC), which are found predominantly Revised: 25 May 2023 in the mucosa of the airways and the gastrointestinal tract, and connective tissue MCs Accepted: 30 May 2023 (CTMC), mainly located in the skin and peritoneal cavity. In these locations, MCs act as Published: 8 June 2023 sentinel cells, initiating immune responses to exogenous stimuli [6–10]. Mast cells play a crucial role in the maintenance of many physiological functions, such as vasodilation, homeostasis, tissue remodeling, angiogenesis, activation, mediator release, Copyright: © 2023 by the authors. and the development and amplification of the inflammatory response and antimicrobial Licensee MDPI, Basel, Switzerland. defense. However, the mechanisms of immunological interactions are still not fully un- This article is an open access article derstood [11–17]. At the same time, MCs are involved in the pathophysiology of multiple distributed under the terms and diseases including allergies, asthma, cardiovascular diseases, pulmonary fibrosis, osteo- conditions of the Creative Commons porosis, autoimmune arthritis, multiple sclerosis, mast cell activation syndromes, neoplastic Attribution (CC BY) license (https:// diseases, inflammatory bowel syndrome, and inflammatory bowel disease (IBD) [14,18–27]. creativecommons.org/licenses/by/ Moreover, in the particular case of IBD, it has been suggested that IL-10 modulates 4.0/). the interaction of the host with the microbiota [28–30]. In this sense, mice deficient in Int. J. Mol. Sci. 2023, 24, 9875. https://doi.org/10.3390/ijms24129875 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 9875 2 of 15 interleukin 10 (IL-10−/− ) spontaneously develop enterocolitis under conventional housing conditions after 12 weeks of age, which is then used as a model of IBD [31–33]. This could be explained by the fact that IL-10 would be a major immunosuppressive and anti- inflammatory factor that is indispensable for the homeostatic control of infection and inflammation [30,34]. Previous studies on the relationship between MCs and IL-10 have shown that IL-10 regulates the proliferation and production of cytokines and the differentiation of MCs [35–37]. In addition, mice that are deficient in both MCs and IL-10 exhibit exacerbated colitis compared to single IL-10 knockout mice, which clearly reinforces the relationship between MCs and IL-10 [26,38]. Considering that MCs are highly heterogeneous according to their origin and location in the body, more information is needed regarding the role of IL-10 in different types of MCs. Simultaneously, as part of their innate immunity, MCs play a pivotal role in inflamma- tion and antimicrobial immunity mediated by pattern recognition receptors (PRRs) and can thus identify both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [39–41]. Via PRRs, MCs can recognize and respond to bacteria and viruses located both extracellularly and intracellularly [24,42–47]. Once activated after PAMP-PRR interactions, MCs release a variety of biologically active mediators, such as proteases, cytokines, and chemokines [25,39,44,48–51]. These mediators induce a diver- sity of biological effects, including the recruitment and activation of inflammatory cells, smooth muscle contractions, and an increase in vascular permeability [8,26,52]. In addition, responses to PRR activation are conditioned by the level of expression of PRRs in different MC subtypes [44]. Therefore, it is necessary to analyze each specific MC subpopulation individually to properly understand their modulation by PRRs. The aim of this study was to characterize the effect of IL-10 deficiency on the MC phenotype and its activation via PRRs in stages before and after the development of colitis in a murine model of IBD. 2. Results 2.1. Morphological Analysis and mMCP-1 and mMCP-6 Characterization of MLMC and PCMC MLMCs and PCMCs displayed typical MC phenotypes. There was no obvious mor- phologic difference between WT and IL-10−/− MCs (Figure S1). The expression of mMCP-1 and mMCP-6 was similar in both genotypes from MLMC, with a higher expression (~1.5 folds) of each at 20 weeks in the WT animals. (Figure 1A,B). In PCMCs, no expression of mMCP-1 was detected, whereas expression of mMCP-6 was similar in both genotypes isolated from 6-week-old mice. However, in knockout PCMCs at 20 weeks, lower mMCP-6 expression was detected compared to age-matched WT (4.65 ± 1.369 vs. 0.405 ± 0.246 folds; p < 0.01) (Figure 1C,D). 2.2. Effects of IL-10 Deletion on TLR2 Expression and Release of Inflammatory Mediators Related to Its Activation by Pam2 CSK4 in MLMC and PCMC No significant differences in TLR2 expression were observed in MLMCs and PCMCs under basal conditions (time zero, unstimulated) for both genotypes (WT and IL-10−/− mice), but TLR2 expression was decreased in 20-week-old compared with 6-week-old mice (−70% WT; −60% IL-10−/− ). When analyzing the effect of IL-10 deletion on TLR2 expression and inflammatory mediator release in MLMCs and PCMCs treated for 6 and 24 h with Pam2 CSK4, respectively, a lower percentage of TLR2+ was observed after stimulation in IL-10−/− cells compared with WT cells (p < 0.05) (Figure 2A,D). Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 16 Int. J. Mol. Sci. 2023, 24, 9875 3 of 15 Figure 1. Gene expression of mMCP-1 and mMCP-6 by MLMCs and PCMCs derived from wild type and IL-10−/− mice. Gene expression of mouse mast cell protease (mMCP)-1 (A,C) and mMCP-6 (B,D) 1. Figure was Gene assessed in mucosal-like expression of mMCP-1 and mast cells (MLMCs; mMCP-6 by MLMCs A,B) and and peritoneum-derived PCMCs cultured derived from wild type mast and IL-10 cells −/− mice. Gene expression of mouse mast cell protease (mMCP)-1 (A,C) and mMCP-6 (B,D) (PCMCs; C,D). Relative expression of mMCPs mRNA was analyzed by quantitative reverse was assessed in mucosal-like transcription-PCR mast cellsto(MLMCs; and normalized transcript A,B) andof levels peritoneum-derived β-2-microglobulin. cultured mast levels Expression cells were (PCMCs; C,D). Relative expression of mMCPs mRNA was analyzed by quantitative reverse tran- measured in wild type (WT; filled circles) and IL-10−/− (empty squares). All the analyses were scription-PCR and normalized to transcript levels of β-2-microglobulin. Expression levels were Int. J. Mol. Sci. 2023, 24, x FOR PEER performed usingtype REVIEW in wild measured cells isolated (WT; filled from 6 (left circles) panels) and IL-10 and 20squares). −/− (empty (right panels) All the week oldwere analyses mice. Data 4per- of 16 are mean ± formed standard using error offrom cells isolated 4–6 6animals per group. (left panels) and 20 ** p < 0.01 (right vs. week panels) the wild type, Data old mice. 2-way areANOVA mean with ± Bonferroni standard error of 4–6 N.D.: posttest. animalsnotper group. ** p < 0.01 vs. the wild type, 2-way ANOVA with Bonfer- detected. roni posttest. N.D.: not detected. 2.2. Effects of IL‐10 Deletion on TLR2 Expression and Release of Inflammatory Mediators Re‐ lated to Its Activation by Pam2CSK4 in MLMC and PCMC No significant differences in TLR2 expression were observed in MLMCs and PCMCs under basal conditions (time zero, unstimulated) for both genotypes (WT and IL-10−/− mice), but TLR2 expression was decreased in 20-week-old compared with 6-week-old mice (−70% WT; −60% IL-10−/−). When analyzing the effect of IL-10 deletion on TLR2 ex- pression and inflammatory mediator release in MLMCs and PCMCs treated for 6 and 24 h with Pam2CSK4, respectively, a lower percentage of TLR2+ was observed after stimula- tion in IL-10−/− cells compared with WT cells (p < 0.05) (Figure 2A,D). Regarding inflammatory cytokine secretion (IL-6 and TNFα), deletion of IL-10 signif- icantly reduced IL-6 secretion at 6 and 20 weeks and TNFα secretion at 20 weeks, in MLMC (Figure 2B,C; p < 0.05), and secretion of both cytokines was lower in knockout PCMC at week 6 (2644 ± 673 vs. 774 ± 367 pg/mL for IL-6; 619 ± 174 vs. 237 ± 105 pg/mL for TNFα) (Figure 2E,F). When comparing both populations of MCs, higher TNFα was observed by PCMCs compared to MLMC (619 ± 174 vs. 136 ± 7 pg/mL for WT; 237 ± 105 vs. 126 ± 15 pg/mL for IL-10−/− at 6 h). Of note, when MLMC and PCMC were subjected to any of the stimuli used in the study, it was not possible to detect at the protein level IL-10, IL-1β, IL-12p70, or IFN-γ. Figure 2. Effects of IL-10 deletion on TLR2 expression and release of inflammatory mediators related to Figure 2. Effects of IL-10 deletion on TLR2 expression and release of inflammatory mediators related its toactivation by Pam its activation 2 CSK4 by Pam in MLMCs and PCMCs. The expression of toll-like receptor 2 (TLR2) was 2CSK4 in MLMCs and PCMCs. The expression of toll-like receptor 2 (TLR2) assesses in mucosal-like was assesses mast cells in mucosal-like mast(MLMCs; A) and A) cells (MLMCs; peritoneum-derived culturedcultured and peritoneum-derived mast cells (PCMCs; mast cells D). (PCMCs; D). The percentage of cells positive for TLR2 was characterized in basal conditions (un- stimulated; time 0) and after the stimulation with synthetic diacylated lipoprotein Pam2CSK4 (PAM). IL-6 (B,E) and TNFα (C,F) levels were measured in wild type (WT; filled circles) and IL-10−/− (empty squares) MLMC (B,C) and PCMC (E,F) left unstimulated or stimulated with PAM. All the analyses were performed using cells isolated from 6 (left panels) and 20 (right panels) week old Int. J. Mol. Sci. 2023, 24, 9875 4 of 15 The percentage of cells positive for TLR2 was characterized in basal conditions (unstimulated; time 0) and after the stimulation with synthetic diacylated lipoprotein Pam2 CSK4 (PAM). IL-6 (B,E) and TNFα (C,F) levels were measured in wild type (WT; filled circles) and IL-10−/− (empty squares) MLMC (B,C) and PCMC (E,F) left unstimulated or stimulated with PAM. All the analyses were performed using cells isolated from 6 (left panels) and 20 (right panels) week old mice. Data are mean ± standard error of 4–6 animals per group. * p < 0.05, ** p < 0.01 vs. wild type, 2-way ANOVA with Bonferroni posttest. Regarding inflammatory cytokine secretion (IL-6 and TNFα), deletion of IL-10 sig- nificantly reduced IL-6 secretion at 6 and 20 weeks and TNFα secretion at 20 weeks, in MLMC (Figure 2B,C; p < 0.05), and secretion of both cytokines was lower in knockout PCMC at week 6 (2644 ± 673 vs. 774 ± 367 pg/mL for IL-6; 619 ± 174 vs. 237 ± 105 pg/mL for TNFα) (Figure 2E,F). When comparing both populations of MCs, higher TNFα was observed by PCMCs compared to MLMC (619 ± 174 vs. 136 ± 7 pg/mL for WT; 237 ± 105 vs. 126 ± 15 pg/mL for IL-10−/− at 6 h). Of note, when MLMC and PCMC were subjected to any of the stimuli used in the study, it was not possible to detect at the protein level IL-10, IL-1β, IL-12p70, or IFN-γ. 2.3. Effects of IL-10 Deletion on TLR4 Expression and Release of Inflammatory Mediators Related to Its Activation by LPS in MLMC and PCMC The basal proportion of MLMC expressing TLR4 on their surface was lower5 in Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW knock- of 16 out mice at 6 weeks (p < 0.001), but this difference was no longer apparent at week 20, becoming similar in both genotypes (~0.6%). In the unstimulated PCMC, no differences were observed at week 6, with TLR4 expression being undetectable at week 20 (Figure 3A,D). a reduction in the proinflammatory response of MLMC to LPS was observed at 20 weeks After TLR4 stimulation with LPS in MLMCs, the differences between both genotypes were compared to 6 weeks (approximately more than 1000 pg/mL for both cytokines). maintained (p < 0.05). As for MLMCs, LPS did not affect TLR4 surface expression in PCMC. Figure 3. Effects of IL-10 deletion on TLR4 expression and release of inflammatory mediators related Figure 3. Effects of IL-10 deletion on TLR4 expression and release of inflammatory mediators related totoits itsactivation activation by by LPS LPS in in MLMCs MLMCs and andPCMCs. PCMCs.TheTheexpression expressionof of toll-like toll-like receptor receptor 4 (TLR4) 4 (TLR4) was was assesses in mucosal-like mast cells (MLMCs; A) and peritoneum-derived cultured mast cells (PCMCs; D). The percentage of cells positive for TLR4 was characterized in basal conditions (un- stimulated; time 0) and after the stimulation with lipopolysaccharide (LPS) from Escherichia coli O55:B5. IL-6 (B,E) and TNFα (C,F) levels were measured in wild type (WT; filled circles) and IL-10−/− (empty squares) MLMCs (B,C) and PCMCs (E,F) left unstimulated or stimulated with LPS. All the Int. J. Mol. Sci. 2023, 24, 9875 5 of 15 assesses in mucosal-like mast cells (MLMCs; A) and peritoneum-derived cultured mast cells (PCMCs; D). The percentage of cells positive for TLR4 was characterized in basal conditions (unstim- ulated; time 0) and after the stimulation with lipopolysaccharide (LPS) from Escherichia coli O55:B5. IL-6 (B,E) and TNFα (C,F) levels were measured in wild type (WT; filled circles) and IL-10−/− (empty squares) MLMCs (B,C) and PCMCs (E,F) left unstimulated or stimulated with LPS. All the analyses were performed using cells isolated from 6 (left panels) and 20 (right panels) week old mice. Data are mean ± standard error of 4–6 animals per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. wild type, 2-way ANOVA with Bonferroni posttest. N.D.: not detected. Regarding the secretion of proinflammatory mediators, TLR4 activation induced the release of IL-6 and TNFα by MLMC but not by PCMC (Figure 3). When both genotypes were compared, MLMC derived from 6-week-old IL-10−/− mice secreted more IL-6 after TLR4 stimulation than WT animals of the same age (Figure 3B; p < 0.05). On the other hand, at week 20, no differences in IL-6 secretion were observed. TNFα secretion by MLMC was not influenced by IL-10 and, although it did not reach statistical significance, it tended to be higher at week 20 in the case of IL-10−/− PCMC (Figure 3C,F). Furthermore, a reduction in the proinflammatory response of MLMC to LPS was observed at 20 weeks compared to 6 weeks (approximately more than 1000 pg/mL for both cytokines). 2.4. Effects of IL-10 Deletion on TLR7 Expression and Release of Inflammatory Mediators Related to Its Activation by Imiquimod in MLMC and PCMC Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW In basal conditions, MLMC derived from 6-week-old mice of both genotypes 6 ofshowed 16 similar levels of TLR7 expression. On the other hand, at week 20, the expression of TLR7 was lower in MLMC when IL-10 was not present (p < 0.001). In contrast to MLMC, TLR7 (107 ±was 13 vs.not 57 detected ± 6 pg/mLin atPCMC isolated 24 h) (Figure from 4B,C). any of Finally, the genotypes consistent with theatlack anyoftime detec-point. tion of TLR7 (Figure 4A,D).inStimulation PCMC, we did not observe of MLMC with any effect of was imiquimod imiquimod on proinflammatory associated with a time-lapse of cytokine TLR7 genesecretion (Figure expression 4E,F). in both genotypes at week 6 (p < 0.001), and lower TLR7 expression in IL-10 − / − cells at 20 weeks (p < 0.001), like that observed in basal conditions. Figure 4. Effects of IL-10 deletion on TLR7 expression and release of inflammatory mediators related to its activation by Imiquimod in MLMCs and PCMCs. Gene expression of toll-like receptor 7 (TLR7) Figure 4. Effects of IL-10 deletion on TLR7 expression and release of inflammatory mediators related to its activation by Imiquimod in MLMCs and PCMCs. Gene expression of toll-like receptor 7 (TLR7) was assesses in mucosal-like mast cells (MLMCs; A) and peritoneum-derived cultured mast cells (PCMCs; D). Relative expression of TLR7 mRNA was analyzed by quantitative reverse transcrip- tion-PCR and normalized to transcript levels of β-2-microglobulin in basal conditions (unstimu- lated; time 0) and after the stimulation with imiquimod (IMQ). IL-6 (B,E) and TNFα (C,F) levels Int. J. Mol. Sci. 2023, 24, 9875 6 of 15 was assesses in mucosal-like mast cells (MLMCs; A) and peritoneum-derived cultured mast cells (PCMCs; D). Relative expression of TLR7 mRNA was analyzed by quantitative reverse transcription- PCR and normalized to transcript levels of β-2-microglobulin in basal conditions (unstimulated; time 0) and after the stimulation with imiquimod (IMQ). IL-6 (B,E) and TNFα (C,F) levels were measured in wild type (WT; filled circles) and IL-10−/− (empty squares) MLMCs (B,C) and PCMCs (E,F) left unstimulated or stimulated with IMQ. All the analyses were performed using cells isolated from 6 (left panels) and 20 (right panels) week old mice. Data are mean ± standard error of 4–6 animals per group. * p < 0.05 and *** p < 0.001 vs. wild type, 2-way ANOVA with Bonferroni post-test. N.D.: not detected. The proinflammatory pattern depended on the genotype of the cells and the age of the animals from which they were isolated. At week 6 and after 24 h of stimulation, IL-6 levels were higher in MLMC obtained from IL-10−/− mice (597 ± 15 vs. 498 ± 20 pg/mL). In contrast, at week 20, TNFα secretion in response to imiquimod was higher in WT MLMC (107 ± 13 vs. 57 ± 6 pg/mL at 24 h) (Figure 4B,C). Finally, consistent with the lack of detection of TLR7 in PCMC, we did not observe any effect of imiquimod on proinflammatory cytokine secretion (Figure 4E,F). 2.5. Effects of IL-10 Deletion on NOD2 Expression and Release of Inflammatory Mediators Related to Its Activation by MDP in MLMC and PCMC The gene expression of the NOD2 receptor was only detected in MLMC isolated from 6-week-old mice. In this case, we observed a downregulation of gene expression in −/− Int. J. Mol. Sci. 2023, 24, x FOR PEERIL-10 REVIEW animals compared with WT mice (p < 0.05). However, these differences were 7 of 16 no longer detected after cell culture and stimulation with MDP, which induced a reduction in NOD2 expression (Figure 5A,D). Figure 5. Effects of IL-10 deletion on NOD2 expression and release of inflammatory mediators related toFigure its activation 5. EffectsbyofMDP IL-10in MLMCs deletion onand NOD2PCMCs. Gene and expression expression release of of nucleotide-binding oligomeriza- inflammatory mediators re- tion lateddomain-containing to its activation by protein MDP in2MLMCs (NOD2)and was assesses PCMCs. in mucosal-like Gene expression ofmast cells (MLMCs;oli- nucleotide-binding A) and peritoneum-derived cultured mast cells gomerization domain-containing (PCMCs; protein D). Relative 2 (NOD2) expression was assesses inofmucosal-like NOD2 mRNA was cells mast analyzed by(MLMCs; A) and quantitative peritoneum-derived reverse cultured transcription-PCR and mast cells (PCMCs; normalized D). Relative to transcript levelsexpression of NOD2 in of β-2-microglobulin mRNA was analyzed by quantitative reverse transcription-PCR and normalized to transcript levels of β-2-microglobulin in basal conditions (unstimulated; time 0) and after the stimulation with mu- ramyldipeptide (MDP). IL-6 (B,E) and TNFα (C,F) levels were measured in wild type (WT; filled circles) and IL-10−/− (empty squares) MLMCs (B,C) and PCMCs (E,F) left unstimulated or stimulated with MDP. All the analyses were performed using cells isolated from 6 (left panels) and 20 (right Int. J. Mol. Sci. 2023, 24, 9875 7 of 15 basal conditions (unstimulated; time 0) and after the stimulation with muramyldipeptide (MDP). IL-6 (B,E) and TNFα (C,F) levels were measured in wild type (WT; filled circles) and IL-10−/− (empty squares) MLMCs (B,C) and PCMCs (E,F) left unstimulated or stimulated with MDP. All the analyses were performed using cells isolated from 6 (left panels) and 20 (right panels) week old mice. Data are mean ± standard error of 4–6 animals per group. * p < 0.05 vs. the wild type, 2-way ANOVA with Bonferroni posttest. N.D.: not detected. In relation to cytokine release, no changes were observed in IL-6 or TNFα levels associated with the stimulation with MDP or with the expression of IL-10 in any MC subtype (Figure 5B,C,E,F). 2.6. Effects of the Lack IL-10 on FcεRI-Mediated Responses in MLMC and PCMC No differences between genotypes were observed in MLMC at 6 weeks; however, FcεRI-mediated IL-6 and TNFα secretion was lower in IL-10−/− cells at 20 weeks (p < 0.01) (Figure 6A,B). In PCMC, IL-10 deletion reduced cytokines levels detected after FcεRI activation at week 6 (p < 0.05). In contrast, when obtained at week 20, IL-10 −/− Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 16 MCs produced similar levels of IL-6 and higher levels of TNFα compared to WT cells (5207 ± 83 vs. 4211 ± 195 pg/mL) (Figure 6B,D). Figure 6. Effects of the lack IL-10 on FcεRI-mediated release of proinflammatory mediators in MLMCs and PCMCs. IL-6 (A,C) and TNFα (B,D) levels were measured in wild type (WT; filled circles) and Figure IL-10 −/− 6.(empty Effectssquares) of the lack IL-10 (A,B) MLMCs on FcεRI-mediated releaseleft and PCMCs (C,D) of unstimulated proinflammatory mediators (NS) in or stimulated MLMCs and PCMCs. IL-6 (A,C) and TNFα (B,D) levels were measured in wild type (WT; filled with dinitrophenyl-human serum albumin. All the analyses were performed using cells isolated circles) and IL-10−/− (empty squares) MLMCs (A,B) and PCMCs (C,D) left unstimulated (NS) or stim- from 6 (left ulated withpanels) and 20 (right serum dinitrophenyl-human week old panels)albumin. Allmice. Data are the analyses mean were ± standard performed erroriso- using cells of the mean latedoffrom 3–4 6animals per group. (left panels) p < 0.05 and 20 *(right panels) ** p