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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2002-03-0921. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-03-0921v1 101/1/210    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by von Stebut, E. Articles by Maurer, M. Search for Related Content PubMed PubMed Citation Articles by von Stebut, E. Articles by Maurer, M. Related Collections Immunobiology Phagocytes Chemokines, Cytokines, and Interleukins Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 January 2003, Vol. 101, No. 1, pp. 210-215 IMMUNOBIOLOGY Early macrophage influx to sites of cutaneous granuloma formation is dependent on MIP-1/ released from neutrophils recruited by mast cell-derived TNF Esther von Stebut, Martin Metz, Genevieve Milon, Jürgen Knop, and Marcus Maurer From the Department of Dermatology, University-Hospital Mainz, Germany; and Unite d'Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, Paris, France.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Macrophages (M) play a crucial role in the development of cutaneous granulomas (CGs) initiated by foreign bodies or invasive microorganisms. However, little is known about how M are recruited to sites of CG formation. To test whether mast cells (MCs) contribute to early M recruitment to developing granulomas, CGs were induced in MC-deficient KitW/KitW-v mice by injection of polyacrylamide gel (PAG). KitW/KitW-v mice as well as mice deficient in the MC product TNF exhibited markedly reduced M numbers in CGs. M recruitment was restored in KitW/KitW-v mice reconstituted with MCs from Kit+/+ or TNF+/+, but not from TNF/ mice. MC-TNF-dependent M influx required prior recruitment of MIP-1/-producing neutrophils (PMNs), as PMN depletion before induction of CGs completely inhibited M influx, which was restored after reconstitution with PMN supernatants. These findings indicate that M recruitment to cutaneous PAG- induced granulomas is the result of a sequence of inflammatory processes initiated by MC-derived TNF followed by PMN influx and MIP-1a/ release. (Blood. 2003;101:210-215) © 2003 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Mononuclear phagocytes (M) are crucial effector cells in the induction of protective cutaneous immune responses to infections by (1) microbes such as Leishmania major and Mycobacterium leprae1 or (2) foreign bodies such as polyacrylamide gel (PAG). M-mediated protection from such intracellular microorganisms includes phagocytosis, recruitment of other proinflammatory leukocytes (ie, neutrophils [PMNs], eosinophils, and lymphocytes),1,2 and, most importantly, development of cutaneous granulomas (CGs) aimed at clearing or restricting the growth of microorganisms at sites of infection.1,3 Failure to recruit M and PMNs in microbial infection results in impaired granuloma formation associated with greatly impaired host defense and systemic disease.4 Despite the great importance of M in granulomatous inflammation,2 little is known about the mechanisms regulating their recruitment to developing cutaneous granulomas. To better characterize the chain of inflammatory processes preceding M-dependent CG formation and to elucidate potential mechanisms involved in M recruitment, we have investigated PAG-induced granulomatous inflammation in the absence of mast cells (MCs). MCs are well known to initiate and orchestrate inflammatory skin responses to allergens and pathogens. We and others have shown that early activation of MCs is necessary for protective immune responses to bacterial infection in the context of acute septic peritonitis.5-8 In this setting, impaired MC activation, such as in complement (C3)-deficient mice, correlates with markedly reduced (1) release of TNF, (2) PMN recruitment, (3) clearance of bacteria, and, consequently, (4) survival.7 Furthermore, MCs have been shown to contribute to acute inflammatory monocyte recruitment by releasing MCP-1 or histamine in allergic asthma as well as after the implantation of biomaterial.9,10 However, the role of MCs in more slowly developing immune responses such as cutaneous granuloma formation has not yet been investigated. Here, we assessed early CG formation in MC-deficient mice, using the model of PAG-induced granulomatous inflammation. We demonstrate that MCs are required to initiate a sequence of inflammatory processes resulting in M recruitment to developing CGs: MC-derived TNF is necessary to recruit PMNs, which control M influx by releasing M-attracting chemokines, including macrophage inflammatory protein-1 (MIP-1) / and MIP-2.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Animals C57BL/6 mice, genetically MC-deficient WBB6F1-KitW/KitW-v (KitW/KitW-v) mice, and congenic wild-type WBB6F1-Kit+/+ (Kit+/+) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). TNF/ mice of a mixed 129/Sv × C57BL/6 genetic background were bred at the Department of Dermatology, Mainz.11 Mice were kept in community cages at the Animal Care Facilities at the University of Mainz. All animal care and experimentation were conducted in accordance with current federal, state, and institutional guidelines. CG model CGs were elicited by subcutaneous or intradermal injection of polyacrylamide gel (PAG) as previously described.12-14 Briefly, PAG was prepared by addition of Biogel P-100 (BioRad, München, Germany) to phosphate buffered saline (PBS; 35 mg/mL). PAG was injected subcutaneously into the neck area in volumes of 1 mL. Initial experiments with C57BL/6 mice confirmed that the cellular infiltrate in  5-day-old granulomas consisted exclusively of PMNs and M as assessed by fluorescence-activated cell-sorter (FACS) analysis.15 In some experiments, depleting anti-mouse neutrophil rat mAb clone NIMP-R14 (100 µg/mouse)16 or clone 7/4 (200 µg/mouse) (Serotec, Hamburg, Germany) or isotype control rat mAb were injected intravenously. Phenotyping of leukocytes recovered from PAG-induced granulomas Leukocytes were harvested from early PAG-induced granulomas and separated from PAG by filtering through a 70-µm filter in cold PBS. Cells were counted, stained for surface Ag expression,14 and analyzed using a FACScan flow cytometer equipped with CellQuest software (Becton Dickinson, Heidelberg, Germany). The antibodies used were as follows: biotin-conjugated F4/80 and fluorescein isothiocyanate (FITC)-conjugated anti-neutrophil mAb (7/4) were obtained from Caltag (Hamburg, Germany); anti-CD16/CD32 (2.4G2), anti-I-Ab (2G9), anti-CD3 (145-2C4), anti-CD4 (L3T4), anti-CD8 (53-6.7), CD11b (M1/70), Gr-1 (RB6-8C5), and anti-NK1.1 (PK136) were purchased from PharMingen (Hamburg, Germany) as biotin- or phycoerythrin (PE)-modified mAb; PE-streptavidin was from Tago (Burlingame, CA). Preparation of MCs MC-deficient skin was reconstituted with peritoneal MCs as both peritoneal and skin MCs are connective tissue type MCs (CTMCs) and share virtually identical phenotypes.8 In brief, the peritoneal cavity was lavaged with 0.9% NaCl, and cell suspensions were stained for MCs by Kimura stain and enumerated. CTMCs were enriched to  95% purity from peritoneal lavage suspension by several gradient centrifugation steps using 23% metrizamide.17 Viability of CTMCs was  90% as assessed by trypan blue exclusion. Cutaneous MC reconstitution of KitW/KitW-v mice The MC deficiency of KitW/KitW-v mice (6-8 weeks old) was corrected selectively and locally by injection of CTMCs.8 CTMCs (1 × 106 in 100 µL 0.9% NaCl) were injected intradermally, and mice were used for experiments, together with sex- and age-matched MC-deficient KitW/KitW-v and Kit+/+ mice, 48 hours after adoptive transfer. MCs were injected into shaved neck skin covering an area of about 1 cm2. Reconstitution of cutaneous MC populations was confirmed by histomorphometric analysis of paraffin-embedded, Giemsa-stained sections of injected skin 48 hours after the injection.8 MC activation assays The extent of MC degranulation in PAG-injected skin was assessed as described previously.18 Biopsies were taken 60 minutes after intradermal injection of 0.1 mL PAG, and sections were examined by an investigator blinded to the experimental design. MC degranulation was assessed by quantitative histomorphometry (at 1000×).18 A minimum of 100 MCs in  5 sections/mouse per treatment group were examined. Measurements of serotonin (5-hydroxytryptamine [5-HT]) release in vitro were performed as previously described.17 Briefly, peritoneal MC suspensions were incubated with 2 µCi (0.074 MBq)[3H]5-HT (Perkin Elmer, Freiburg, Germany) for 2 hours at 37°C, washed, stimulated with PAG or PBS for 15 minutes, and the percentage of total 5-HT release was calculated. Reconstitution of CGs with PMN supernatants and RNAse protection assay PMNs were isolated from 6- and 12-hour-old granulomas by filter separation. PMNs were further enriched by negative depletion of M using a 2-hour plastic adherence step; > 99% of cells in PMN preparations routinely showed positive antineutrophil (clone 7/4) staining as demonstrated by FACS analysis. Chemokine production was determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Wiesbaden, Germany) in 18-hour supernatants of 2 × 106 PMN/mL RPMI/5% fetal calf serum (FCS) supplemented with glutamine, penicillin/streptomycine, and nonessential amino acids.14 PMN supernatants for reconstitution experiments were generated by plating 5 × 106 PMN/mL in RPMI/5% FCS in 6-well plates, and cell-free supernatants were harvested after 48 hours. As a control, RPMI/5% FCS was treated similarly. Supernatants were assayed by ELISA for their chemokine content and were stored at 20°C until used for reconstitution experiments. Biogel granulomas were reconstituted locally twice daily with either media alone or PMN supernatants (0.5 mL/granuloma) or PMN supernatants that had been preincubated with 10 µg/mL anti-MIP-1 and 0.1 µg/mL anti-MIP-1 (R&D Systems) for 30 minutes at 20°C (Ab amounts were about 4-fold of a concentration calculated to inhibit approximately 50% of the activity found in the supernatants). For analysis of chemokine expression, RNA from PMNs isolated from 6- and 12-hour granulomas was generated using the High Pure RNA Isolation Kit from Roche (Mannheim, Germany). Expression of chemokines known to be potent M attractants was assessed following the manufacturer's protocol using the RiboQuant RNAse Protection System and template set mCK5, both obtained from PharMingen. Statistical analysis All data were tested for statistical significance using the unpaired 2-tailed Student t test or 2 test (MC degranulation in situ) and are presented as means ± SEM.     Results Top Abstract Introduction Materials and methods Results Discussion References Recruitment of M during early granuloma formation is MC-dependent M recruitment to early CGs induced by subcutaneous injection of PAG was greatly reduced in genetically MC-deficient KitW/KitW-v mice compared with Kit+/+ mice during the first 72 hours following CG induction (Figure 1A). M numbers in CGs of MC-deficient skin were reduced by 66% (0.3 ± 0.1 vs 0.9 ± 0.1 × 106, P = .001) and 46% (3.5 ± 0.6 vs 6.6 ± 0.7 × 106, P = .004) at 12 hours and 72 hours, respectively, compared with skin of wild-type mice (Figure 1B). To ensure that this defect is due to a lack of MCs in these mice, we reconstituted the skin of KitW/KitW-v mice with Kit+/+ CTMCs (1 × 105) prior to the induction of granulomas, which fully restored the influx of inflammatory M to CGs after 12 hours (Figure 1B). These data suggest that MCs are required for normal recruitment of M to sites of CG formation. View larger version (44K): [in this window] [in a new window]   Figure 1. Early M recruitment to PAG-induced cutaneous granulomas is MC dependent. The absence of MCs in KitW/KitW-v mice results in impaired recruitment of M to early CGs developing at sites of subcutaneous delivery of PAG. (A) Time course of M recruitment to CGs after PAG injection in Kit+/+ and KitW/KitW-v mice. Data were pooled from 8 or more mice per genotype and time point-tested in at least 4 independent experiments. (B) Migration of M to granulomas in KitW/KitW-v mice 12 hours after induction is repaired by reconstitution of the dermis with connective tissue-type MCs obtained from Kit+/+ mice. Data were pooled from 8 or more mice per genotype in 2 independent experiments. Data in panels A and B are shown as means ± SEMs (× 106 cells/granuloma). In 1A and 1B, *P < .05, **P < .005, ***P < .001. (C) Characteristic surface staining with anti-M F4/80 and anti-PMN 7/4 mAb of leukocytes recovered from 24- and 72-hour-old granulomas of normal Kit+/+ mice revealed 2 distinct populations, namely PMNs (7/4+) and M (F4/80+). Numbers reflect F4/80+/7/4 cells and 7/4+/F4/80 cells in percent of total cells. One experiment representative of at least 8 independent analyses is shown. M from developing granulomas were readily identified as F4/80+ and 7/4 cells (Figure 1C). In both MC-deficient KitW/KitW-v mice and normal Kit+/+ mice, M influx to CGs was detected as early as 12 hours after PAG injection. Interestingly, the surface phenotype of infiltrating M (eg, presence of major histocompatibility complex class I and II, or costimulatory molecules) was not affected by the absence of MCs (data not shown). Recruitment of M to skin granulomas is dependent on MC-derived TNF To identify the mechanisms by which MCs control M recruitment to early CGs, we first determined whether CG formation after injection of PAG is associated with MC activation. PAG-injected skin exhibited significantly more extensively and moderately degranulated MCs than vehicle-treated or uninjected skin (P < .001) 1 hour after induction of granulomas in C57BL/6 mice (Figure 2A). PAG was also found to induce substantial degranulation of isolated and highly purified CTMCs in vitro as assessed by serotonin release assays (Figure 2B), suggesting the MCs may recruit M to developing CGs by releasing preformed proinflammatory mediators. View larger version (39K): [in this window] [in a new window]   Figure 2. Induction of cutaneous granuloma formation by PAG depends on release of TNF by MCs. (A) MCs in the direct vicinity of early PAG-induced CGs exhibit signs of profound degranulation. As assessed by quantitative histomorphometry, the extent of MC degranulation 1 hour after PAG injection in C57BL/6 mice was scored as "none" (< 10% of granules exhibited staining alterations and/or exteriorization), "moderate" (10%-50%), or "extensive" (> 50%), and expressed as means ± SEMs (data pooled from 5 mice). (B) Unpurified (purity about 1%) and purified (purity > 95%) peritoneal MCs of C57BL/6 mice release serotonin after incubation with PAG. Data were pooled from 3 independent experiments and expressed as means ± SEMs. (C) TNF-deficient mice exhibit reduced recruitment of M to sites of CG formation after injection of PAG compared with wild-type mice. Data were pooled from 5 or more mice per genotype and time point in 3 independent experiments. (D) Migration of M to granulomas in KitW/KitW-v mice 12 hours after induction is repaired only by reconstitution of the skin with connective tissue-type MCs obtained from TNF+/+ mice, but not after reconstitution with MCs from TNF-deficient mice. Data were pooled from 8 or more mice per genotype in 2 independent experiments. Data in all panels are shown as means ± SEMs. *P < .05, **P < .005, ***P < .001. Since one such MC product, TNF, has been shown to regulate the recruitment of inflammatory cells, including M,19-22 we assessed PAG-induced CG formation in TNF/ mice and TNF+/+ mice. M recruitment to CGs was dramatically impaired in TNF/ mice as compared to TNF+/+ mice (Figure 2C). Notably, up to 7-fold more M per granuloma were recovered in TNF+/+ mice as compared to TNF/ mice (8.9 ± 1.3 vs 1.3 ± 0.7 × 106 at 72 hours, P < .001). Since TNF is not exclusively produced by MCs, but MCs are the only cell type known to contain preformed TNF,19,23 we reconstituted the skin of KitW/KitW-v mice with MCs derived from either TNF+/+ mice or TNF/ mice, thus generating mice that differed solely in containing MCs that could or could not release TNF.7,24 KitW/KitW-v mice showed normal migration of M to PAG-induced granulomas only after reconstitution with TNF+/+ MCs (Figure 2D). Reconstitution with MCs derived from TNF/ mice did not increase M influx, indicating that release of TNF from MCs is required for normal M recruitment to sites of CG formation. M influx to CGs is dependent on PMNs recruited by MC-TNF M migration to inflamed skin is preceded by the influx of large amounts of PMNs, proinflammatory cells that have been shown to migrate to sites of TNF release from MCs and to produce M-recruiting chemokines.25 To assess whether MC-TNF recruits M during early CG formation directly or by inducing immigration of M-attracting PMNs, we first assessed the kinetics of PMN recruitment to PAG-induced granulomas in MC-, TNF-, or MC-TNF-deficient skin. Genetically MC-deficient KitW/KitW-v mice exhibited markedly reduced recruitment of PMNs as compared to wild-type mice after induction of CGs by PAG injection (Figure 3A). PMN numbers in 6- to 72-hour-old CGs were significantly and up to 70% lower as compared to wild-type mice at all time points studied. The absence of MCs did not affect the time course of PMN influx. Adoptive transfer of CTMCs to MC-deficient skin prior to injection of PAG restored normal recruitment of PMNs to CG (Figure 3B). PMN numbers also were greatly reduced in TNF-deficient mice as compared to TNF+/+ mice in up to 5-day-old granulomas (Figure 3C). PMN recruitment was completely restored in KitW/KitW-v mice reconstituted with TNF+/+ MCs, while reconstitution with TNF-deficient MCs did not correct impaired PMN influx in these mice. These observations indicate that MC-derived TNF contributes not only to M recruitment, but also to PMN recruitment to sites of CG formation. View larger version (35K): [in this window] [in a new window]   Figure 3. Neutrophil recruitment to PAG-induced granulomas is dependent on MC-derived TNF. (A) Time course of PMN influx after injection of PAG in Kit+/+ and KitW/KitW-v mice. Data were pooled from 10 or more mice per genotype and time point-tested in at least 7 independent experiments. (B) Impaired recruitment of PMNs to CGs in KitW/KitW-v mice is MC dependent. The dermis of KitW/KitW-v mice was reconstituted with connective tissue-type MCs from normal Kit+/+ mice before PAG injection, and PMN numbers were determined 12 hours after injection of PAG. Data were pooled from 8 or more mice per genotype in at least 4 independent experiments. (C) PMN recruitment in cutaneous granuloma formation is strongly dependent on the presence of TNF. Numbers of infiltrating PMNs were determined in PAG-injected skin of mice deficient in TNF and in wild-type mice. Data were pooled from 5 or more mice per genotype and time point in 3 independent experiments. (D) PMN recruitment was fully restored in skin of TNF+/+ MC-reconstituted KitW/KitW-v mice. Before PAG injection, the dermis of KitW/KitW-v mice was reconstituted with MCs from TNF+/+ mice or TNF-deficient mice, and numbers of PMNs were assessed 12 hours thereafter. Data were pooled from 8 or more mice per genotype in 2 independent experiments. All data in Figure 3 are shown as means ± SEMs (× 106 cells/granuloma). For all panels in Figure 3, *P < .05, ***P < .005, ***P < .001. To determine if MC- and TNF-dependent recruitment of PMNs is involved in the regulation of M recruitment to CGs, C57BL/6 mice were depleted of PMNs by a single intravenous injection of the mAb NIMP-R14 or mAb 7/4 1-3 hours before induction of CG. NIMP-R14 has been shown to eliminate PMNs from the peripheral blood and bone marrow of mice for 2-3 days without affecting other leukocyte populations of the myeloid lineages.16 PMNs were found to be virtually absent in PAG-induced CGs after injection of NIMP-R14, whereas treatment with the isotype control mAb had no effect compared with untreated animals (Figure 4A). Notably, the numbers of F4/80+ M were reduced by 88% and 93% at 24 hours and 72 hours, respectively, after depletion of PMN (Figure 4B), indicating that PMN recruitment is essential for normal M migration to sites of CG formation. View larger version (33K): [in this window] [in a new window]   Figure 4. PMN influx is required for M recruitment during early cutaneous granuloma formation. C57BL/6 mice pretreated with PMN-depleting mAb NIMP-R14 (intravenously, 100 µg, 1 hour) exhibit virtually no recruitment of M to sites of PAG-induced CG formation compared with control Ab-treated mice. Numbers of PMNs (A) and M (B) per CG were assessed 24 hours and 72 hours after injection of PAG. Data were pooled from 5 or more mice per treatment group and time point in 2 independent experiments. Data are shown as means ± SEMs (106 cells/granuloma). ***P < .001. (C) PMNs were isolated from CGs 12 hours after injection of biogel and were stained for anti-neutrophil Ab clone 7/4 (black area, anti-neutrophil Ab; white area, isotype control). Purity of isolated PMNs was determined to be > 99%; one experiment of at least 5 is shown. In panel D, groups of 3 C57BL/6 mice were depleted from PMNs using mAb 7/4 (100 µg intravenously, at 6 hours and +18 hours), and CGs were reconstituted locally twice daily with 0.5 mL/granuloma of either vehicle alone or PMN supernatant (PMN-SPNT). M numbers were determined after 48 hours and are expressed as means ± SEMs (106 cells/granuloma). **P < .005. Data are representative of 3 independent experiments. Soluble mediators released by PMNs rather than structural/molecular changes induced by PMN migration may regulate M recruitment to CGs. To test this hypothesis, we reconstituted CGs in PMN-depleted mice with supernatant derived from PMNs or media alone (Figure 4D). Supernatants from PMNs were generated in RPMI/5% FCS for 48 hours, and purity of isolated PMNs was determined to be > 99% using FACS analysis and staining with anti-neutrophil Ab 7/4 (Figure 4C). Local injection of PMN supernatants twice daily restored > 60% of M influx to sites of CG formation, whereas vehicle alone (media + FCS) did not repair M influx at all. MIP-1/ released by PMNs are responsible for M recruitment to CGs To identify the soluble mediators released by PMNs, we analyzed cytokine release and chemokine expression of PMNs isolated from either 6- or 12-hour-old granulomas (Figure 5A). While we found no significant production of TNF or other cytokines (interleukin-4 [IL-4], IL-12p40, interferon- [IFN]) by PMNs when assessing the supernatants by ELISA (data not shown), RNAse protection assays revealed strong expression of MIP-1, MIP-1, and MIP-2 by PMNs. The C-C chemokines MIP-1 and MIP-1 are strong chemotactic signals for M (compare Luster26), whereas MIP-2, a C-x-C chemokine and the murine homolog for IL-8, is known to recruit PMNs themselves as well as T lymphocytes.26 Therefore, our data suggest that PMN-derived MIP-1 and MIP-1 are responsible for M recruitment to CGs. We next assayed 18-hour-old supernatants from PMNs for protein content by ELISA (Figure 5B). PMNs isolated from CGs predominantly released MIP-1, although we also detected some MIP-1 activity in the supernatants. Both chemokines were up-regulated in TNF-treated PMNs within 18 hours (Figure 5B). Finally, we attempted to inhibit the activity of MIP-1/ in PMN-derived supernatants used for reconstitution experiments by preabsorption with neutralizing antibodies. The 48-hour PMN supernatants used for reconstitution experiments contained 3.7 ± 1.9 pg/mL MIP-1 and 197.4 ± 47 pg/mL MIP-1, whereas RPMI/5% FCS alone was negative for the chemokines by ELISA. As demonstrated in Figure 5C, reconstitution of CGs with PMN supernatants treated with anti-MIP-1 and anti-MIP-1 inhibited about 60% of supernatant-induced recruitment of M to CGs. View larger version (25K): [in this window] [in a new window]   Figure 5. MIP-1/ released by PMNs is responsible for M influx into cutaneous granulomas. (A) Expression of the chemokines MIP-1, MIP-1, and MIP-2 by PMNs from early CGs (6 hours and 12 hours old) was found using the RNAse protection assay (PharMingen). One of 2 experiments with similar results is shown. Lane 1 shows the template set; lanes 2 and 3, PMN RNA; lane 4, control RNA provided by the manufacturer. (B) Production of MIP-1 and MIP-1 was determined in supernatants of 2 × 106 PMNs in fully supplemented media after 18 hours. Chemokine production was enhanced in TNF-treated PMNs (n = 5). In panel C, groups of 3 C57BL/6 mice were depleted of PMNs using mAb 7/4 as shown in Figure 4D, and CGs were reconstituted locally twice daily with either vehicle alone or PMN supernatant (PMN-SPNT) or PMN-SPNT that was pretreated with anti-MIP-1 and anti-MIP-1 for 30 minutes. M numbers were determined after 48 hours and expressed as means ± SEMs (106 cells/granuloma). *P < .05. Data are pooled from 6 mice per group from 2 independent experiments.     Discussion Top Abstract Introduction Materials and methods Results Discussion References MCs are ideally suited to initiate protective immune responses against invading pathogens because (1) MCs are preferentially located at host/environment interfaces (upper dermis in the skin, gut lamina propria, airways' subepithelium), (2) they produce a large array of proinflammatory mediators, many of which are stored within cytoplasmic granules and are released within minutes after activation, and (3) MCs have receptors that can recognize microbial molecules (including complement receptors toll-like receptors, and CD48) and can, therefore, be activated by pathogens via multiple mechanisms, including fimbrial proteins, toxins, and the proteolytic fragments of complement components.19 Indeed, MCs have been reported to be required for the induction of immediate host defense reactions in various models of acute bacterial infections.5-7 Here, we show for the first time that MCs also are required to elicit normal chronic and more slowly developing cellular responses. Indeed, the normal succession of key events in slowly developing immune responses such as CG formation is induced by and dependent on initial MC activation. Specifically, early MC degranulation is needed to elicit recruitment of M, a hallmark feature of granulomatous inflammation (compare Murray3), which is required for the development of CGs. Our findings suggest that MCs facilitate normal chronic granulomatous inflammatory processes by inducing the following chain of events: release of TNF from MCs promotes influx of PMN, which release M-recruiting chemokines (such as MIP-1/, MIP-2), which in turn result in the recruitment of M. The early steps of granuloma development are tightly modeled in leukocyte recruitment to lesions in cutaneous leishmaniasis, where the initial inflammatory phase of granuloma development is characterized by the sequential influx of PMNs, eosinophils, and M to parasite-loaded skin.2 In leishmaniasis, extensive dermal MC degranulation is found at sites of early infection,27 and MCs coincubated with Leishmania major in vitro reportedly release preformed TNF within minutes,28 suggesting that MCs and MC-derived products may modulate the host response to Leishmania, especially during the initial phase of infection. Our data support this hypothesis by showing that MCs contribute significantly to the local inflammatory cutaneous reaction that develops at sites of CG formation in response to foreign bodies such as PAG. In future studies we will investigate in more detail if and how MCs modulate local inflammatory responses observed after intradermal delivery of Leishmania major. Most of the leukocyte recruitment to CGs that we observed was TNF dependent, stressing the essential role that TNF plays in skin inflammation. To our surprise, the absence of TNF resulted in reduced recruitment of M as early as 12 hours after injection of PAG, suggesting that TNF is, at least in part, released from preformed stores. Since MCs are the only resident skin cells shown to contain considerable amounts of preformed TNF, it is mostly likely that most of the TNF that accounts for the initial influx of PMNs and M is released by MCs, although several other types of skin cells are capable of producing TNF.29 At later time points after injection of PAG, the difference in inflammation between MC-deficient and normal wild-type mice was less dramatic, perhaps reflecting TNF release or production of other factors by cells other than MCs (including dendritic cells, resident dermal M, keratinocytes). Most of the MC effect on leukocyte recruitment was TNF-dependent and did not depend on release of other MC mediators since only reconstitution with TNF-producing MCs fully restored inflammatory cell influx to CGs in KitW/KitW-v mice, while the adoptive transfer of TNF-deficient MCs did not improve PMNs or M recruitment in these mice. This observation is supported by a recent report showing that inflammatory skin reactions, including PMN influx in contact hypersensitivity, are impaired in KitW/KitW-v mice and that these responses were normalized only after adoptive transfer of TNF-competent, but not TNF-deficient, MCs.24 In addition, Zhang et al provided evidence that, in vivo, MCs produce the TNF that augments PMN emigration in the context of acute peritoneal inflammation.30 Another remarkable finding of this study was that depletion of PMNs resulted in a strong impairment of M recruitment to the site of CG formation. To ensure that the mononuclear cell population was not affected by PMN depletion, we used anti-neutrophil Ab clone NIMP-R14 for our experiments.16 In agreement with the findings of Tacchini-Cottier et al,16 we observed a similar and overlapping staining pattern of the monoclonal anti-neutrophil antibodies NIMP-R14 and 7/4 on PMNs derived from C57BL/6 mice. No expression of NIMP-R14 or 7/4 by resident or recently extravasated M was detected (Figure 1C and data not shown). The sequential accumulation of first PMNs and then M to tissues after initiation of inflammatory processes is a phenomenon that is observed frequently in a variety of diseases and experimental models, but the strict dependence of M recruitment on prior PMN influx has not been previously demonstrated. Several reports describe impaired M functions and granuloma formation in patients with neutrophil dysfunction or deficiency (such as agranulocytosis), for example, impaired granuloma formation in a patient with severe granulocytopenia led to insufficient control of fungi with severe dissemination of Aspergillus.4 In contrast, in chronic granulomatous disease, hyperactivation of neutrophils and increased release of chemoattractants for M and T cells from PMNs might contribute to poorly controlled inflammatory responses and unstructured granuloma formation in affected patients.31 However, further studies are warranted to better characterize the correlation between PMNs and M recruitment in cutaneous granulomatous development and to elucidate underlying mechanisms. In summary, we show that TNF release from activated MCs regulates PMN influx to sites of granulomatous inflammation, which in turn recruit M to developing cutaneous granulomas by releasing potent M-recruiting chemokines, including MIP-1 and MIP-1. Our studies confirm and extend recently established concepts regarding the role of MCs in host-immune responses. In addition to the sentinel role in acute host-defense reactions against bacteria, MCs may also be critical effector cells responsible for the initiation and orchestration of long-lasting cell-mediated immunity.     Acknowledgments The authors wish to thank Dr George Kollias for kindly providing TNF-deficient mice; Drs Mark C. Udey, Yasmine Belkaid, and Helmut Jonuleit for helpful discussions; Drs Michael Stassen and Edgar Schmitt for help with RNAse protection assays; Drs Kerstin Steinbrink, Karsten Mahnke, and Thomas Tüting for critically reading the manuscript; and Elena Wiese for excellent technical assistance.     Footnotes Submitted March 25, 2002; accepted August 6, 2002. Prepublished online as Blood First Edition Paper, August 15, 2002; DOI 10.1182/blood-2002-03-0921. Parts of this work were supported by grants from the Deutsche Forschungsgemeinschaft (DFG; Ste 833/4-1 and Ma 1909/4-1) and the Mainzer Forschungsförderungsprogramm (MAIFOR) program to E. von S. and M.M. 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