Foreign Body Response to Subcutaneous Implants in Diabetic Rats

Implantation of synthetic matrices and biomedical devices in diabetic individuals has become a common procedure to repair and/or replace biological tissues. However, an adverse foreign body reaction that invariably occurs adjacent to implant devices impairing their function is poorly characterized in the diabetic environment. We investigated the influence of this condition on the abnormal tissue healing response in implants placed subcutaneously in normoglycemic and streptozotocin-induced diabetes in rats. In polyether-polyurethane sponge discs removed 10 days after implantation, the components of the fibrovascular tissue (angiogenesis, inflammation, fibrogenesis, and apoptosis) were assessed. Intra-implant levels of hemoglobin and vascular endothelial growth factor were not different after diabetes when compared with normoglycemic counterparts. However, there were a lower number of vessels in the fibrovascular tissue from diabetic rats when compared with vessel numbers in implants from non-diabetic animals. Overall, the inflammatory parameters (neutrophil accumulation - myeloperoxidase activity, tumor necrosis factor alpha, and monocyte chemotactic protein-1 levels and mast cell counting) increased in subcutaneous implants after diabetes induction. However, macrophage activation (N-acetyl-β-D-glucosaminidase activity) was lower in implants from diabetic rats when compared with those from normoglycemic animals. All fibrogenic markers (transforming growth factor beta 1 levels, collagen deposition, fibrous capsule thickness, and foreign body giant cells) decreased after diabetes, whereas apoptosis (TUNEL) increased. Our results showing that hyperglycemia down regulates the main features of the foreign body reaction induced by subcutaneous implants in rats may be relevant in understanding biomaterial integration and performance in diabetes.     

The role of neutrophil myeloperoxidase in the development of inflammation induced by thermal skin burns

Luminol-dependent chemiluminescence (CL) of blood neutrophils stimulated with phorbol-12- myristate-13-acetate (PMA) and myeloperoxidase (MPO) activity of neutrophils and plasma have been investigated in children (n = 16) during the early period (1?7 days) after thermal skin burns exceeding 20% of total body surface. The CL level of stimulated neutrophils was higher in burn patients than in healthy children of the reference group (p lt; 0.01). Increased neutrophil MPO activity was found in 40% of patients, while increased plasma MPO activity was detected in 57% of patients. The albumin fraction isolated from plasma of burn patients increased the PMA-stimulated CL response of blood from healthy donors. These results suggest that the acute inflammatory response to the thermal burn causes neutrophil activation and MPO release into plasma. MPO-mediated modification of plasma proteins, particularly albumin, may stimulate neutrophil activation and provoke further inflammatory response of the body to the thermal injury.     

The neutrophil response to polyvinyl sponge implantation

Neutrophil release and migration in mice were studied over a 24-hr period after the sc implantation of a single polyvinyl sponge. The release of neutrophils from the marrow was evaluated by directly counting the residual neutrophils in the femoral marrow of animals with sponges. Sponge and tissue neutrophil content was determined by extraction and assay of myeloperoxidase (MPO), a marker enzyme for neutrophils. A maximum depletion of 48% of the mature neutrophils in the marrow was observed 5 hr after sponge implantation, in keeping with significant release of neutrophils for migration to the sponge. The released cells were not found in the circulating granulocyte pool, since neutropenia was noted. The accumulation of neutrophils in the sponge increased throughout the 24-hr period, whereas in the tissue adjacent to the sponge maximum accumulation of neutrophils occurred within 7 hr. In fact, neutrophils migrated to at least three sites--the sponge, the skin overlying the sponge, and the skin in which an incision had been made to insert the sponge. The sponge content of neutrophils represented 0.3-33% of the neutrophils migrating to the combined lesion (sponge and skin sites). Therefore, if the neutrophil response to foreign body implantation is to be measured in its entirety, it is necessary to quantify not only the neutrophils within the foreign body but also those in the tissues surrounding it. These studies describe an animal model for neutrophil release and migration to tissues following a standard stimulus. It is proposed that this model may be useful in exploring the factors which influence the release and migration of neutrophils in vivo.     

Anti-Inflammatory Effects of IL-27 in Zymosan-Induced Peritonitis: Inhibition of Neutrophil Recruitment Partially Explained by Impaired Mobilization from Bone Marrow and Reduced Chemokine Levels

Rapid activation of the innate immune system is critical for an efficient host response to invading pathogens. However, the inflammatory reaction has to be strictly controlled to minimize harmful immunopathology. A number of mediators including the cytokine interleukin-27 (IL-27) appear to be responsible for limitation and resolution of inflammation. Despite increasing knowledge of its suppressive effects on T cells, the influence on neutrophils and macrophages is poorly understood. To determine the role of IL-27 in innate immune responses we analysed the effect of IL-27 in a T cell independent model of zymosan-induced peritonitis. Early administration of recombinant IL-27 strongly reduced the number of neutrophils recruited to the peritoneal cavity after zymosan application as well as the neutrophil frequency in the blood. Simultaneously, IL-27 reduced the release of neutrophils from the bone marrow upon inflammation. Although cytokine levels were not affected by IL-27 treatment, the levels of the chemokines KC, MCP-1 and MIP-1α in the peritoneal fluid were strongly decreased. These findings demonstrate that IL-27 is able to control mobilisation and recruitment of neutrophils into the peritoneal cavity and identify a novel mechanism to limit inflammation caused by innate immune cells.     

Role of hypothermia induced by tumor necrosis factor on apoptosis and function of inflammatory neutrophils in mice

Changes in body temperature and cell infiltration, mediated by cytokines including tumor necrosis factor-alpha (TNF-alpha), occur during inflammation, but a role of body temperature on inflammatory responses remains obscure. Intraperitoneal injection of 10% casein to mice resulted in transient hypothermia followed by neutrophil accumulation in peritoneal cavities. Peritoneal TNF-alpha was rapidly raised, and pretreatment of mice with an anti-TNF-alpha antibody promoted temperature restoration and partially inhibited neutrophil accumulation. To investigate direct effects of body temperature on neutrophils, peritoneal or peripheral blood neutrophils were cultured at 35 degrees C or 37 degrees C with or without recombinant murine TNF-alpha (100 ng/ml) or a protein synthesis inhibitor cycloheximide (1 microg/ml). Significant inhibition of spontaneous and TNF-alpha-induced apoptosis was obtained at 35 degrees C compared with 37 degrees C, an effect that was not altered by the addition of cycloheximide. Moreover, phagocytic ability of peritoneal neutrophils was significantly enhanced by incubating them at the lower temperature. These results indicate that mild hypothermia induced by endogenous TNF-alpha has enhancing roles on neutrophil survival and function during peritoneal inflammation.     

Heat Shock Modulates Neutrophil Motility in Zebrafish

Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.     

Differential Dependencies of Monocytes and Neutrophils on Dectin-1, Dectin-2 and Complement for the Recognition of Fungal Particles in Inflammation

We have re-investigated the role of the complement system and the non-opsonic pattern recognition receptors dectin-1 and dectin-2 in the recognition of fungal particles by inflammatory neutrophils, monocytes and macrophages. We have used in vivo and ex vivo models to study the recognition and response of these cells: i) We confirm previous observations regarding the importance of complement to neutrophil but not monocytic responses; ii) We show that dectin-1 is important for driving inflammatory cell recruitment to fungal stimuli and that it biases the immediate inflammatory response to one that favors neutrophil over monocyte recruitment; iii) We show that dectin-2 contributes to the physical recognition of fungal particles by inflammatory monocytes/macrophages, but is also expressed on neutrophils, where we show it has the potential to contribute to cellular activation; iv) Additionally, we show that serum-opsonization has the potential to interfere with non-opsonic recognition of fungal particles by dectin-1 and dectin-2, presumably through masking of ligands. Collectively these roles are consistent with previously described roles of dectin-1 and dectin-2 in driving inflammatory and adaptive immune responses and complement in containing fungal burdens. This study emphasizes the importance of heterogeneity of receptor expression across myeloid cell subsets in protective immune responses.     

Human Neutrophil Cytoskeletal Dynamics and Contractility Actively Contribute to Trans-Endothelial Migration

Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks transmigration, thus establishing a critical role for neutrophil actin dynamics in transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which “opens the door” in the endothelium and facilitates transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the transmigration step of the immune response.     

Interleukin 10 modulation of neutrophil subsets infiltrating lungs during Streptococcus pneumoniae infection

Interleukin-10 production and lung neutrophil infiltration are two essential components of the balanced immune response to pneumonia caused by Streptococcus pneumoniae. Here we describe the existence of two neutrophil subsets in lungs during experimental S. pneumoniae infection in mice, which have different size, granularity and expression of activation markers. During infection, both neutrophils subsets were increased in the lungs of IL-10 producing mice, however this increment was significantly higher in the absence of this cytokine. These results suggest that IL-10 is a key cytokine that regulates lung inflammation during bacterial infection caused by specific neutrophil subsets infiltrating the lungs.     

2-Undecanone derived from Pseudomonas aeruginosa modulates the neutrophil activity

Pseudomonas aeruginosa (P. aeruginosa) is a well-known Gram-negative opportunistic pathogen. Neutrophils play key roles in mediating host defense against P. aeruginosa infection. In this study, we identified a metabolite derived from P. aeruginosa that regulates neutrophil activities. Using gas chromatography-mass spectrometry, a markedly increased level of 2-undecanone was identified in the peritoneal fluid of P. aeruginosa-infected mice. 2-Undecanone elicited the activation of neutrophils in a G_ai-phospholipase C pathway. However, 2-undecanone strongly inhibited responses to lipopolysaccharide and bactericidal activity of neutrophils against P. aeruginosa by inducing apoptosis. Our results demonstrate that 2-undecanone from P. aeruginosa limits the innate defense activity of neutrophils, suggesting that the production of inhibitory metabolites is a strategy of P. aeruginosa for escaping the host immune system.     

Schistosoma japonicum Egg Specific Protein SjE16.7 Recruits Neutrophils and Induces Inflammatory Hepatic Granuloma Initiation

Neutrophils are known to play a major role in the egg granulomatous lesions caused by Schistosoma japonicum, but the precise mechanism by which eggs recruit or active neutrophil is unknown. Here we report S. japonicum egg specific EF-hand protein-SjE16.7 is a potent neutrophil recruiter and initiates the egg associated inflammatory granuloma in schistosomiasis. We show that the expression of SjE16.7 at level of both mRNA and protein is restricted to the egg stage. It locates in the miracidium and subshell area of the egg and can be secreted by the egg. The antigenic properties of SjE16.7 strongly suggest a role for SjE16.7 as an egg-derived molecule involved in host-parasite interactions. To study SjE16.7 functions in vivo, we challenged murine air pouch with recombinant SjE16.7. The results showed SjE16.7 trigged more inflammatory cell infiltration than vehicle or control protein. Using peritoneal exudate neutrophils from mice, we found that SjE16.7 significantly induced neutrophil chemotaxis in vitro, and the observed phenotypes were associated with enhanced Rac GTPase activation in SjE16.7 treated cells. Finally, in vivo hepatic granuloma formation model showed SjE16.7 coupled beads recruited more inflammatory cell infiltration than control beads. Our findings suggest SjE16.7 is an important pathogenic factor derived from egg. By recruiting neutrophils and inducing local inflammation, SjE16.7 facilitates eggs to be excreted through gut tissues and also initiates pathology in the liver; therefore SjE16.7 is a possible target for the prevention and treatment of schistosomiasis.     

IL-1α modulates neutrophil recruitment in chronic inflammation induced by hydrocarbon oil

Exposure to naturally occurring hydrocarbon oils is associated with the development of chronic inflammation and a wide spectrum of pathological findings in humans and animal models. The mechanism underlying the unremitting inflammatory response to hydrocarbons remains largely unclear. The medium-length alkane 2,6,10,14 tetramethylpentadecane (also known as pristane) is a hydrocarbon that potently elicits chronic peritonitis characterized by persistent infiltration of neutrophils and monocytes. In this study, we reveal the essential role of IL-1α in sustaining the chronic recruitment of neutrophils following 2,6,10,14 tetramethylpentadecane treatment. IL-1α and IL-1R signaling promote the migration of neutrophils to the peritoneal cavity in a CXCR2-dependent manner. This mechanism is at least partially dependent on the production of the neutrophil chemoattractant CXCL5. Moreover, although chronic infiltration of inflammatory monocytes is dependent on a different pathway requiring TLR-7, type I IFN receptor, and CCR2, the adaptor molecules MyD88, IL-1R-associated kinase (IRAK)-4, IRAK-1, and IRAK-2 are shared in regulating the recruitment of both monocytes and neutrophils. Taken together, our findings uncover an IL-1α-dependent mechanism of neutrophil recruitment in hydrocarbon-induced peritonitis and illustrate the interactions of innate immune pathways in chronic inflammation.     

Foreign body giant cell induction in the CSF-1-deficient osteopetrotic (op/op) mouse

The osteopetrosis (op) mutation in mice is characterized by generalized skeletal sclerosis; reduced numbers of osteoclasts, macrophages, and monocytes; and failure to be cured by bone marrow transplantation. This mutation has been shown to result from an absence of colony-stimulating factor-1 (CSF-1) and reported to be cured by treatment with CSF-1. Macrophage polykaryons are known to be formed by fusion of mononuclear precursors and the presence of subcutaneous implants can elicit the formation of macrophage polykaryons. In order to determine if recruitment of foreign body giant cells is also impaired in osteopetrotic mice, tissue reactions to subcutaneously implanted polyvinyl sponges were studied and compared with normal mice. Our result showed that, in the op mouse, recruitment of macrophages and foreign body giant cells in response to the implants was quantitatively not different from that of normal mice. However, these cells were smaller and did not migrate as deeply into the implant as those seen in normal littermates. In contrast, resident macrophages obtained by peritoneal lavage were significantly reduced in op mice. These data indicate that there is a deficiency in the ability of op mice to mount a foreign body giant cell response to an implanted sponge characterized by a deficiency in the recruitment of precursor cells that are capable of either full development and spreading or migration into the implanted sponge. These data add to the emerging appreciation of the regional differences among macrophage populations in their dependence on CSF-1 for differentiation and survival.     

Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling

The early inflammatory response to influenza virus infection contributes to severe lung disease and continues to pose a serious threat to human health. The mechanisms by which neutrophils gain entry to the respiratory tract and their role during pathogenesis remain unclear. Here, we report that neutrophils significantly contributed to morbidity in a pathological mouse model of influenza virus infection. Using extensive immunohistochemistry, bone marrow transfers, and depletion studies, we identified neutrophils as the predominant pulmonary cellular source of the gelatinase matrix metalloprotease (MMP) 9, which is capable of digesting the extracellular matrix. Furthermore, infection of MMP9-deficient mice showed that MMP9 was functionally required for neutrophil migration and control of viral replication in the respiratory tract. Although MMP9 release was toll-like receptor (TLR) signaling-dependent, MyD88-mediated signals in non-hematopoietic cells, rather than neutrophil TLRs themselves, were important for neutrophil migration. These results were extended using multiplex analyses of inflammatory mediators to show that neutrophil chemotactic factor, CCL3, and TNFα were reduced in the Myd88 ^−/− airways. Furthermore, TNFα induced MMP9 secretion by neutrophils and blocking TNFα in vivo reduced neutrophil recruitment after infection. Innate recognition of influenza virus therefore provides the mechanisms to induce recruitment of neutrophils through chemokines and to enable their motility within the tissue via MMP9-mediated cleavage of the basement membrane. Our results demonstrate a previously unknown contribution of MMP9 to influenza virus pathogenesis by mediating excessive neutrophil migration into the respiratory tract in response to viral replication that could be exploited for therapeutic purposes.     

Real-time dynamics of neutrophil clustering in response to phototoxicity-induced cell death and tissue damage in mouse ear dermis

ABSTRACT

Neutrophils are highly motile innate immune cells; they actively migrate in response to inflammatory signals. Using two-photon intravital microscopy, we discovered that neutrophils form stable clusters upon phototoxicity at a certain threshold. Without significant damage to the collagen structure of mouse dermis, neutrophils aggregated together with nearby neutrophils. Surprisingly, this in situ neutrophil clustering resulted in rigorous changes of migratory direction. The density of residing neutrophils was also a critical factor affecting clustering. Additionally, we found that the triggering point of neutrophil aggregation was correlated with the structure of the extracellular matrix in the ear dermis, where autofluorescence was strongly observed. This swarming behavior of neutrophils may reflect an unknown communication mechanism of neutrophils during migration under sterile injury.     

Pertussis Toxin Stimulates IL-17 Production in Response to Bordetella pertussis Infection in Mice

In a mouse model of respiratory tract infection by Bordetella pertussis, bacteria multiply in the airways over the first week and are then cleared over the next 3–4 weeks by the host immune response. Pertussis toxin (PT), a virulence factor secreted exclusively by B. pertussis, promotes bacterial growth in the airways by suppression and modulation of host immune responses. By comparison of wild type and PT-deficient strains, we examined the role of PT in modulating airway cytokine and chemokine responses affecting neutrophil recruitment during B. pertussis infection in mice. We found that, despite early inhibition of neutrophil recruitment by PT, high numbers of neutrophils were recruited to the airways by 4 days post-infection with the wild type strain, but not with the PT-deficient strain, and that this correlated with upregulation of neutrophil-attracting chemokine gene expression. In addition, there was similar upregulation of genes expressing the cytokines IL-17A (IL-17), TNF-α and IFN-γ, indicating a mixed Th1/Th17 response. Expression of IL-6, a cytokine involved in Th17 induction, was upregulated earlier than the IL-17 response. We showed that PT, rather than bacterial numbers, was important for induction of these responses. Flow cytometric analysis revealed that the IL-17-producing cells were macrophages and neutrophils as well as T cells, and were present predominantly in the airways rather than the lung tissue. Antibody neutralization of IL-17 significantly reduced chemokine gene expression and neutrophil recruitment to the airways, but only modestly increased peak bacterial loads. These data indicate that PT stimulates inflammatory responses by induction of Th1- and Th17-associated cytokines, including IL-17, during B. pertussis infection in mice, but a role for IL-17 in protection against the infection remains to be established.     

Function and regulation of the neutrophil MEL-14 antigen in vivo: comparison with LFA-1 and MAC-1

The CD11/18 (LFA-1, Mac-1) molecules participate in neutrophil adhesion to cultured endothelium in vitro and are critical for effective neutrophil localization into inflamed tissues in vivo. More recently, the MEL-14 Ag, which was first defined as a lymphocyte homing receptor, has also been implicated in inflammatory neutrophil extravasation. Here we compare the regulation and function of these adhesion molecules on neutrophils during the in vivo inflammatory response. The MEL-14 Ag is expressed at high levels on bone marrow and peripheral blood neutrophils, but is lost on neutrophils isolated from the thioglycollate-inflamed peritoneal cavity. In contrast, Mac-1 is up-regulated on inflammatory neutrophils and little change is seen in the level of LFA-1 expression. In vitro activation of bone marrow neutrophils with PMA or leukotriene B4 results in a dose dependent increase in Mac-1 and decrease in MEL-14 Ag expression within 1 h after treatment, thus reflecting what is found during inflammation in vivo. Neutrophils activated in vitro or in vivo (MEL-14Low, Mac-1Hi) do not home to inflammatory sites in vivo, correlating with the loss of the MEL-14 Ag and the increased Mac-1 expression. Anti-LFA-1, anti-Mac-1, or MEL-14 antibody given i.v. suppress neutrophil accumulation within the inflamed peritoneum (38%, 30%, and 37% of medium control, respectively) without affecting the levels of circulating neutrophils. However, when FITC-labeled cells are precoated with the mAb and injected i.v., only MEL-14 inhibits extravasation into the inflamed peritoneum (25% of medium control). Finally, in ex vivo adhesion assays of neutrophil binding to high endothelial venules in inflamed-lymph node frozen sections MEL-14 inhibits greater than 90%. anti-LFA-1 20 to 30% and anti-Mac-1 less than 10% of the binding of bone marrow neutrophils to inflamed-lymph node high endothelial venules. These results confirm that both the MEL-14 antigen and Mac-1/LFA-1 are important in neutrophil localization to inflamed sites in vivo, but suggest that their roles in endothelial cell interactions are distinct.     

Leukocyte activation by malarial pigment

Malarial pigment, a unique hemozoin crystal composed of unit cells of heme dimers, is present in large amounts in circulating monocytes and neutrophils and can persist unchanged in macrophages for several months. In the present study, we investigated the effect of hemozoin not only on macrophages, but also on neutrophils. We used beta-hematin (BH), a chemically synthetic crystal structurally identical to hemozoin, for these studies. In vitro, BH up-regulated the expression of tumor necrosis factor-alpha in whole blood and in isolated peritoneal macrophages, indicating that hemozoin is able to stimulate monocytes. BH stimulated murine peritoneal neutrophils to express macrophage inflammatory protein-2 (MIP-2), a homologue of human interleukin-8 that is used as a marker of neutrophil activation. Injecting BH into the peritoneal cavity resulted in a dose-dependent migration of neutrophils and a high level of myeloperoxidase activity of peritoneal cells. Finally, BH directly induced neutrophil chemotaxis in vitro. Taken together, these results suggest that the malarial pigment hemozoin can activate leukocytes and may participate in the pathology of severe malaria.     

Type I interferon signaling regulates the composition of inflammatory infiltrates upon infection with Listeria monocytogenes

Type I IFN is key to the immune response to viral pathogens, however its role in bacterial infections is less well understood. Mice lacking the type I IFN receptor (IFNAR-/-) demonstrate enhanced resistance to infection with Listeriamonocytogenes. We have now determined that following infection with Listeria, the composition of innate cells recruited to the peritoneal cavity of IFNAR-/- mice reflects an increase in the frequency of neutrophils and a decrease in monocyte frequency compared to WT controls. These differences in inflammatory infiltrates could not be attributed to distinct bone marrow composition prior to infection or to level of apoptosis. We also observed no differences in neutrophil oxidative burst. However, blocking CXCR2 prevented enhanced neutrophil influx and hampered bacterial clearance. Taken together, these studies highlight a novel mechanism by which type I interferon signaling regulates the immune response to Listeria, through negative regulation of chemokines driving neutrophil recruitment.