Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia.

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

DNA-binding protein-A promotes kidney ischemia/reperfusion injury and participates in mitochondrial function.

DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.

Role of Mast-Cell-Derived RANKL in Ovariectomy-Induced Bone Loss in Mice.

Mast cells may contribute to osteoporosis development, because patients with age-related or post-menopausal osteoporosis exhibit more mast cells in the bone marrow, and mastocytosis patients frequently suffer from osteopenia. We previously showed that mast cells crucially regulated osteoclastogenesis and bone loss in ovariectomized, estrogen-depleted mice in a preclinical model for post-menopausal osteoporosis and found that granular mast cell mediators were responsible for these estrogen-dependent effects. However, the role of the key regulator of osteoclastogenesis, namely, receptor activator of NFκB ligand (RANKL), which is secreted by mast cells, in osteoporosis development has, to date, not been defined. Here, we investigated whether mast-cell-derived RANKL participates in ovariectomy (OVX)-induced bone loss by using female mice with a conditional Rankl deletion. We found that this deletion in mast cells did not influence physiological bone turnover and failed to protect against OVX-induced bone resorption in vivo, although we demonstrated that RANKL secretion was significantly reduced in estrogen-treated mast cell cultures. Furthermore, Rankl deletion in mast cells did not influence the immune phenotype in non-ovariectomized or ovariectomized mice. Therefore, other osteoclastogenic factors released by mast cells might be responsible for the onset of OVX-induced bone loss.

Mast Cells Trigger Disturbed Bone Healing in Osteoporotic Mice.

Mast cells are important tissue-resident sensor and effector immune cells but also play a major role in osteoporosis development. Mast cells are increased in numbers in the bone marrow of postmenopausal osteoporotic patients, and mast cell-deficient mice are protected from ovariectomy (OVX)-induced bone loss. In this study, we showed that mast cell-deficient Mcpt5-Cre R-DTA mice were protected from OVX-induced disturbed fracture healing, indicating a critical role for mast cells in the pathomechanisms of impaired bone repair under estrogen-deficient conditions. We revealed that mast cells trigger the fracture-induced inflammatory response by releasing inflammatory mediators, including interleukin-6, midkine (Mdk), and C-X-C motif chemokine ligand 10 (CXCL10), and promote neutrophil infiltration into the fracture site in OVX mice. Furthermore, mast cells were responsible for reduced osteoblast and increased osteoclast activities in OVX mice callus, as well as increased receptor activator of NF-κB ligand serum levels in OVX mice. Additional in vitro studies with human cells showed that mast cells stimulate osteoclastogenesis by releasing the osteoclastogenic mediators Mdk and CXCL10 in an estrogen-dependent manner, which was mediated via the estrogen receptor alpha on mast cells. In conclusion, mast cells negatively affect the healing of bone fractures under estrogen-deficient conditions. Hence, targeting mast cells might provide a therapeutic strategy to improve disturbed bone repair in postmenopausal osteoporosis. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Nitric oxide controls proliferation of Leishmania major by inhibiting the recruitment of permissive host cells.

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.

Metabolic Interdependency of Th2 Cell-Mediated Type 2 Immunity and the Tumor Microenvironment.

The function of T cells is critically dependent on their ability to generate metabolic building blocks to fulfil energy demands for proliferation and consecutive differentiation into various T helper (Th) cells. Th cells then have to adapt their metabolism to specific microenvironments within different organs during physiological and pathological immune responses. In this context, Th2 cells mediate immunity to parasites and are involved in the pathogenesis of allergic diseases including asthma, while CD8+ T cells and Th1 cells mediate immunity to viruses and tumors. Importantly, recent studies have investigated the metabolism of Th2 cells in more detail, while others have studied the influence of Th2 cell-mediated type 2 immunity on the tumor microenvironment (TME) and on tumor progression. We here review recent findings on the metabolism of Th2 cells and discuss how Th2 cells contribute to antitumor immunity. Combining the evidence from both types of studies, we provide here for the first time a perspective on how the energy metabolism of Th2 cells and the TME interact. Finally, we elaborate how a more detailed understanding of the unique metabolic interdependency between Th2 cells and the TME could reveal novel avenues for the development of immunotherapies in treating cancer.

ATP/IL-33-triggered hyperactivation of mast cells results in an amplified production of pro-inflammatory cytokines and eicosanoids.

IL-33 and ATP are alarmins, which are released upon damage of cellular barriers or are actively secreted upon cell stress. Due to high-density expression of the IL-33 receptor T1/ST2 (IL-33R), and the ATP receptor P2X7, mast cells (MCs) are one of the first highly sensitive sentinels recognizing released IL-33 or ATP in damaged peripheral tissues. Whereas IL-33 induces the MyD88-dependent activation of the TAK1-IKK2-NF-κB signalling, ATP induces the Ca2+ -dependent activation of NFAT. Thereby, each signal alone only induces a moderate production of pro-inflammatory cytokines and lipid mediators (LMs). However, MCs, which simultaneously sense (co-sensing) IL-33 and ATP, display an enhanced and prolonged activation of the TAK1-IKK2-NF-κB signalling pathway. This resulted in a massive production of pro-inflammatory cytokines such as IL-2, IL-4, IL-6 and GM-CSF as well as of arachidonic acid-derived cyclooxygenase (COX)-mediated pro-inflammatory prostaglandins (PGs) and thromboxanes (TXs), hallmarks of strong MC activation. Collectively, these data show that co-sensing of ATP and IL-33 results in hyperactivation of MCs, which resembles to MC activation induced by IgE-mediated crosslinking of the FcεRI. Therefore, the IL-33/IL-33R and/or the ATP/P2X7 signalling axis are attractive targets for therapeutical intervention of diseases associated with the loss of integrity of cellular barriers such as allergic and infectious respiratory reactions.

S-Layer From Lactobacillus brevis Modulates Antigen-Presenting Cell Functions via the Mincle-Syk-Card9 Axis.

C-type lectin receptors (CLRs) are pattern recognition receptors that are crucial in the innate immune response. The gastrointestinal tract contributes significantly to the maintenance of immune homeostasis; it is the shelter for billions of microorganisms including many genera of Lactobacillus sp. Previously, it was shown that host-CLR interactions with gut microbiota play a crucial role in this context. The Macrophage-inducible C-type lectin (Mincle) is a Syk-coupled CLR that contributes to sensing of mucosa-associated commensals. In this study, we identified Mincle as a receptor for the Surface (S)-layer of the probiotic bacteria Lactobacillus brevis modulating GM-CSF bone marrow-derived cells (BMDCs) functions. We found that the S-layer/Mincle interaction led to a balanced cytokine response in BMDCs by triggering the release of both pro- and anti-inflammatory cytokines. In contrast, BMDCs derived from Mincle-/-, CARD9-/- or conditional Syk-/- mice failed to maintain this balance, thus leading to an increased production of the pro-inflammatory cytokines TNF and IL-6, whereas the levels of the anti-inflammatory cytokines IL-10 and TGF-ß were markedly decreased. Importantly, this was accompanied by an altered CD4+ T cell priming capacity of Mincle-/- BMDCs resulting in an increased CD4+ T cell IFN-γ production upon stimulation with L. brevis S-layer. Our results contribute to the understanding of how commensal bacteria regulate antigen-presenting cell (APC) functions and highlight the importance of the Mincle/Syk/Card9 axis in APCs as a key factor in host-microbiota interactions.

Directional mast cell degranulation of tumor necrosis factor into blood vessels primes neutrophil extravasation.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Mast Cells Occupy Stable Clonal Territories in Adult Steady-State Skin.

Mast cells (MCs) are tissue-resident hematopoietic cells intensely studied for their role as effectors in allergic immune responses. Yolk sac-derived embryonic MCs first populate tissues and are later replaced by definitive MCs. We show that definitive MC progenitors expand locally in skin and form clonal colonies that cover stable territories. In MC-deficient skin, colonies grow by proliferation of MCs at the border of the clonal territory. Clonal growth ceases at common borders of neighboring colonies. In steady state, colony self-renewal is independent of bone marrow contribution, and the clonal architecture remains fixed if not disturbed by skin inflammation. Inflammatory cues increase MC density setpoint, stimulating the influx of new progenitors from the bone marrow as well as proliferation of skin-resident cells. The expanding new arrivals disrespect territories of preexisting MC clones. We conclude that during a limited window early in development, definitive MC precursors efficiently enter the skin, expand, and self-maintain, occupying stable territories. In adulthood, circulating progenitors, excluded from steady-state skin, are recruited only into inflamed skin where they clonally expand alongside proliferating skin-resident MCs, disorganizing the original architecture of clonal territories.

The Role of Mast Cells in Bone Metabolism and Bone Disorders.

Mast cells (MCs) are important sensor and effector cells of the immune system that are involved in many physiological and pathological conditions. Increasing evidence suggests that they also play an important role in bone metabolism and bone disorders. MCs are located in the bone marrow and secrete a wide spectrum of mediators, which can be rapidly released upon activation of mature MCs following their differentiation in mucosal or connective tissues. Many of these mediators can exert osteocatabolic effects by promoting osteoclast formation [e.g., histamine, tumor necrosis factor (TNF), interleukin-6 (IL-6)] and/or by inhibiting osteoblast activity (e.g., IL-1, TNF). By contrast, MCs could potentially act in an osteoprotective manner by stimulating osteoblasts (e.g., transforming growth factor-ß) or reducing osteoclastogenesis (e.g., IL-12, interferon-γ). Experimental studies investigating MC functions in physiological bone turnover using MC-deficient mouse lines give contradictory results, reporting delayed or increased bone turnover or no influence depending on the mouse model used. By contrast, the involvement of MCs in various pathological conditions affecting bone is evident. MCs may contribute to the pathogenesis of primary and secondary osteoporosis as well as inflammatory disorders, including rheumatoid arthritis and osteoarthritis, because increased numbers of MCs were found in patients suffering from these diseases. The clinical observations could be largely confirmed in experimental studies using MC-deficient mouse models, which also provide mechanistic insights. MCs also regulate bone healing after fracture by influencing the inflammatory response toward the fracture, vascularization, bone formation, and callus remodeling by osteoclasts. This review summarizes the current view and understanding of the role of MCs on bone in both physiological and pathological conditions.

Mast cells as protectors of health.

Mast cells (MCs), which are well known for their effector functions in TH2-skewed allergic and also autoimmune inflammation, have become increasingly acknowledged for their role in protection of health. It is now clear that they are also key modulators of immune responses at interface organs, such as the skin or gut. MCs can prime tissues for adequate inflammatory responses and cooperate with dendritic cells in T-cell activation. They also regulate harmful immune responses in trauma and help to successfully orchestrate pregnancy. This review focuses on the beneficial effects of MCs on tissue homeostasis and elimination of toxins or venoms. MCs can enhance pathogen clearance in many bacterial, viral, and parasitic infections, such as through Toll-like receptor 2-triggered degranulation, secretion of antimicrobial cathelicidins, neutrophil recruitment, or provision of extracellular DNA traps. The role of MCs in tumors is more ambiguous; however, encouraging new findings show they can change the tumor microenvironment toward antitumor immunity when adequately triggered. Uterine tissue remodeling by α-chymase (mast cell protease [MCP] 5) is crucial for successful embryo implantation. MCP-4 and the tryptase MCP-6 emerge to be protective in central nervous system trauma by reducing inflammatory damage and excessive scar formation, thereby protecting axon growth. Last but not least, proteases, such as carboxypeptidase A, released by FcεRI-activated MCs detoxify an increasing number of venoms and endogenous toxins. A better understanding of the plasticity of MCs will help improve these advantageous effects and hint at ways to cut down detrimental MC actions.

Unimpaired Responses to Vaccination With Protein Antigen Plus Adjuvant in Mice With Kit-Independent Mast Cell Deficiency.

Innate inflammatory responses are crucial for induction and regulation of T cell and antibody responses. Mast cell (MC)-deficient Kit mutant mice showed impaired adaptive immunity, suggesting that MCs provide essential adjuvant activities, and pharmacological MC activation was proposed as a new adjuvant principle. However, the Kit mutations result in complex alterations of the immune system in addition to MC deficiency. We revisited the role of MCs in vaccination responses using Mcpt5-Cre R26DTA/DTA and Cpa3Cre/+ mice that lack connective tissue MCs or all MCs, respectively, but feature an otherwise normal immune system. These animals showed no impairment of T and B cell responses to intradermal vaccination with protein antigen plus complete Freund's adjuvant. Moreover, we demonstrate that the adjuvant effects of the MC secretagogue c48/80 in intradermal or mucosal immunization are independent of the presence of MCs. We hence find no evidence for a regulation by MCs of adaptive immune responses to protein antigens. The finding that immunological MC functions differ from those suggested by experiments in Kit mutants, emphasizes the importance of rigorous tests in Kit-independent MC-deficiency models.

The Neurobeachin-like 2 Protein Regulates Mast Cell Homeostasis.

The neurobeachin-like 2 protein (Nbeal2) belongs to the family of beige and Chediak-Higashi (BEACH) domain proteins. Loss-of-function mutations in the human NBEAL2 gene or Nbeal2 deficiency in mice cause gray platelet syndrome, a bleeding disorder characterized by macrothrombocytopenia, splenomegaly, and paucity of α-granules in megakaryocytes and platelets. We found that in mast cells, Nbeal2 regulates the activation of the Shp1-STAT5 signaling axis and the composition of the c-Kit/STAT signalosome. Furthermore, Nbeal2 mediates granule formation and restricts the expression of the transcription factors, IRF8, GATA2, and MITF as well as of the cell-cycle inhibitor p27, which are essential for mast cell differentiation, proliferation, and cytokine production. These data demonstrate the relevance of Nbeal2 in mast cells above and beyond granule biosynthesis.

Membrane-bound stem cell factor is the major but not only driver of fibroblast-induced murine skin mast cell differentiation.

The maintenance and modulation of cutaneous mast cell (MC) numbers is held to be important for skin immune responses to allergens and pathogens. The increase in MC numbers in the skin is achieved by proliferation and the differentiation of precursor to mature MCs. Fibroblast-derived SCF is thought to be the major skin MC growth factor and it potently induces MC proliferation. The mechanisms of fibroblast-induced skin MC differentiation, including the role of SCF, however, remain insufficiently characterized and understood. Using cocultures of immature murine MCs and fibroblasts, we found that the adhesion of immature MCs to fibroblasts via VCAM-1 and α4 ß7 integrin is very important for subsequent differentiation, which is driven by fibroblast membrane-bound SCF and additional fibroblast-derived membrane-bound signals. Thus, our results show that fibroblast-induced MC differentiation is induced by direct cell-cell contact and involves both Kit-dependent and Kit-independent pathways. Our findings add to the understanding of how immature mast cells mature in murine skin and encourage further analyses of the underlying mechanisms, which may result in novel targets for the modulation of skin mast cell driven diseases.

TAK1 and IKK2, novel mediators of SCF-induced signaling and potential targets for c-Kit-driven diseases.

NF-κB activation depends on the IKK complex consisting of the catalytically active IKK1 and 2 subunits and the scaffold protein NEMO. Hitherto, IKK2 activation has always been associated with IκBα degradation, NF-κB activation, and cytokine production. In contrast, we found that in SCF-stimulated primary bone marrow-derived mast cells (BMMCs), IKK2 is alternatively activated. Mechanistically, activated TAK1 mediates the association between c-Kit and IKK2 and therefore facilitates the Lyn-dependent IKK2 activation which suffices to mediate mitogenic signaling but, surprisingly, does not result in NF-κB activation. Moreover, the c-Kit-mediated and Lyn-dependent IKK2 activation is targeted by MyD88-dependent pathways leading to enhanced IKK2 activation and therefore to potentiated effector functions. In neoplastic cells, expressing constitutively active c-Kit mutants, activated TAK1 and IKKs do also not induce NF-κB activation but mediate uncontrolled proliferation, resistance to apoptosis and enables IL-33 to mediate c-Kit-dependent signaling. Together, we identified the formation of the c-Kit-Lyn-TAK1 signalosome which mediates IKK2 activation. Unexpectedly, this IKK activation is uncoupled from the NF-κB-machinery but is critical to modulate functional cell responses in primary-, and mediates uncontrolled proliferation and survival of tumor-mast cells. Therefore, targeting TAK1 and IKKs might be a novel approach to treat c-Kit-driven diseases.

Mast-Cell-Derived TNF Amplifies CD8(+) Dendritic Cell Functionality and CD8(+) T Cell Priming.

Mast cells are critical promoters of adaptive immunity in the contact hypersensitivity model, but the mechanism of allergen sensitization is poorly understood. Using Mcpt5-CreTNF(FL/FL) mice, we show here that the absence of TNF exclusively in mast cells impaired the expansion of CD8(+) T cells upon sensitization and the T-cell-driven adaptive immune response to elicitation. T cells primed in the absence of mast cell TNF exhibited a diminished efficiency to transfer sensitization to naive recipients. Specifically, mast cell TNF promotes CD8(+) dendritic cell (DC) maturation and migration to draining lymph nodes. The peripherally released mast cell TNF further critically boosts the CD8(+) T-cell-priming efficiency of CD8(+) DCs, thereby linking mast cell effects on T cells to DC modulation. Collectively, our findings identify the distinct potential of mast cell TNF to amplify CD8(+) DC functionality and CD8(+) T-cell-dominated adaptive immunity, which may be of great importance for immunotherapy and vaccination approaches.

Neutrophils are required for both the sensitization and elicitation phase of contact hypersensitivity.

Allergic contact dermatitis and its animal model, contact hypersensitivity (CHS), are T cell-mediated inflammatory skin diseases induced by contact allergens. Though numerous cellular and molecular players are known, the mechanism of chemical-induced sensitization remains poorly understood. Here, we identify neutrophils as crucial players in the sensitization phase of CHS. Genetic deficiency of neutrophils caused by myeloid-specific deletion of Mcl-1 or antibody-mediated depletion of neutrophils before sensitization abrogated the CHS response. Neutrophil deficiency reduced contact allergen-induced cytokine production, gelatinase release, and reactive oxygen species production in naive mice. Mast cell deficiency inhibited neutrophil accumulation at the site of sensitization. In turn, neutrophils were required for contact allergen-induced release of further neutrophil-attracting chemokines, migration of DCs to the draining lymph nodes, and priming of allergen-specific T cells. Lymph node cells from mice sensitized in the absence of neutrophils failed to transfer sensitization to naive recipients. Furthermore, no CHS response could be induced when neutrophils were depleted before elicitation or when normally sensitized lymph node cells were transferred to neutrophil-deficient recipients, indicating an additional role for neutrophils in the elicitation phase. Collectively, our data identify neutrophils to be critically involved in both the sensitization and elicitation phase of CHS.

Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.

Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4-dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.