Intramuscular injection of hrRANTES causes mast cell recruitment and increased transcription of histidine decarboxylase in mice: lack of effects in genetically mast cell-deficient W/WV mice

RANTES (regulated upon activation, normal T cell expressed and presumably secreted) and other chemoattractant proteins are members of the intercrine or chemokine family of proinflammatory basic polypeptides. RANTES is a prototype of the C-C chemokine subfamily that acts as a selective chemoattractant for human monocytes and CD4-positive lymphocytes and increases the adherence of monocytes to endothelial cells. However, the role of RANTES in white cells is still unclear. We report here that hrRANTES at 20 ng/50 microl in mice causes mast cell recruitment 4 h after intramuscular injection, an effect inhibited by anti-RANTES, as evidenced by 0.1% Toluidine blue, a specific dye for coloring mast cells. Injections of PBS (50 microl) vehicle (negative control) did not produce any appreciable inflammatory response, whereas injection of lipopolysaccharide 20 ng/50 microl (positive control) generated a marked inflammatory state. When RANTES was injected intramuscularly in genetically mast cell-deficient W/Wv mice, the inflammatory effect was not present. The RANTES injection sites were then excised and studied under an optical and electron microscope. A Northern blot analysis was performed using a probe that was prepared to detect mRNA encoding the histidine decarboxylase (HDC) gene on excised muscle tissue. We found that hrRANTES provoked generation of HDC mRNA from muscle tissue after 4 h. These effects were inhibited by an anti-RANTES antibody and were absent in genetically mast cell-deficient mice. The increasing number of mast cells in the RANTES injection sites led to an augmentation of histamine content compared to controls (PBS). The injection of hrRANTES 20 ng/20 microl into the sole of a rat paw confirmed the inflammatory and the mast cell recruitment potential of this chemokine. In these studies, hrRANTES injections in muscle tissue provided direct in vivo evidence that RANTES has a significant effect on mast cell recruitment and HDC mRNA generation.     

In vivo and in vitro responses to poly(ethylene terephthalate-co-diethylene glycol terephthalate) and polyethylene oxide blends

Although biocompatible polymeric compounds are generally nontoxic, nonimmunogenic, and chemically inert, implants made from these materials may trigger acute and chronic inflammatory responses. These inflammatory reactions may induce degeneration of implanted biopolymer. Interactions between implanted biomaterial and inflammatory cells are mediated by many cellular events involving cellular adhesion and activation. We studied the inflammatory responses in vivo and in vitro to samples of biopolymers composed of poly(ethylene terephthalate-co-diethylene glycol terephthalate) plus 0, 5, 25% of polyethylene oxide. We observed that these biopolymers did not induce inflammatory responses when implanted in the peritoneal cavity of mice for 28 days. However we observed deposition of hyaluronic acid at the surface of implanted biomaterial, suggesting that tolerance to biomaterial occurred after surgical implantation. No significant adhesion of inflammatory cells such as mononuclear phagocytes and peripheral leukocytes were observed in vitro, when poly(ethylene terephthalate-co-diethylene glycol terephthalate) blends were used as substratum to cellular adhesion. These results suggest that blends composed of poly(ethylene terephthalate-co-diethylene glycol terephthalate) induce low inflammatory cell adhesion, since no rejection of biopolymer was observed when implanted in experimental animal models.     

Prognostic impact of morphometric nuclear grade of endometrial carcinoma

BACKGROUND & AIMS: The pathogenesis of Clostridium difficile toxin A-induced intestinal inflammation is not completely understood. The aim of this study was to define the contribution of mast cells to the fluid secretion and neutrophil infiltration associated with toxin A-induced enteritis. METHODS: Fluid secretion and neutrophil infiltration in toxin A- or buffer-challenged ileal loops were assessed in normal, mast cell-deficient, and mast cell-deficient KitW/KitW-v mice that had undergone selective repair of their mast cell deficiency. The effect of a specific substance P-receptor antagonist was also studied. RESULTS: Intestinal fluid secretion and neutrophil recruitment were significantly diminished in mast cell-deficient KitW/KitW-v and mast cell-deficient MgfSl/MgfSl-d mice compared with the respective normal mice. Mast cell-reconstituted KitW/KitW-v mice showed responses similar to the normal congenic mice. Administration of a specific substance P-receptor antagonist (CP-96,345) reduced toxin A-induced intestinal fluid secretion and inhibited neutrophil infiltration in normal, mast cell-deficient KitW/KitW-v, and mast cell-reconstituted KitW/KitW-v mice. CONCLUSIONS: C. difficile toxin A elicits intestinal fluid secretion and neutrophil infiltration by both mast cell-dependent and -independent pathways, and substance P participates in both pathways.     

Induction of a selective and persistent extravasation of neutrophils into the peritoneal cavity by tryptase mouse mast cell protease 6

Recombinant mouse mast cell protease 6 (mMCP-6) was generated to study the role of this tryptase in inflammatory reactions. Seven to forty-eight hours after the i.p. injection of recombinant mMCP-6 into BALB/c, mast cell-deficient WCB6F1-Sl/Sl(d), C5-deficient, or mMCP-5-null mice, the number of neutrophils in the peritoneal cavity of each animal increased significantly by >50-fold. The failure of the closely related recombinant tryptase mMCP-7 to induce a comparable peritonitis indicates that the substrate specificities of the two tryptases are very different. Unlike most forms of acute inflammation, the mMCP-6-mediated peritonitis was relatively long lasting and neutrophil specific. Mouse MCP-6 did not induce neutrophil chemotaxis directly in an in vitro assay, but did promote chemotaxis of the leukocyte in the presence of endothelial cells. Mouse MCP-6 did not induce cultured human endothelial cells to express TNF-alpha, RANTES, IL-1alpha, or IL-6. However, the tryptase induced endothelial cells to express large amounts of IL-8 continually over a 40-h period. Neither enzymatically active mMCP-7 nor enzymatically inactive pro-mMCP-6 was able to induce endothelial cells to increase their expression of IL-8. Although the mechanism by which mMCP-6 induces neutrophil accumulation in tissues remains to be determined, the finding that mMCP-6 induces cultured human endothelial cells to selectively release large amounts of IL-8 raises the possibility that this tryptase regulates the steady state levels of neutrophil-specific chemokines in vivo during mast cell-mediated inflammatory events.     

Mast cell-dependent neutrophil and mononuclear cell recruitment in immunoglobulin E-induced gastric reactions in mice

BACKGROUND & AIMS: Immunoglobulin (Ig) E-dependent reactions elicit an immediate response and can also result in a late-phase reaction that is characterized by the infiltration of leukocytes. This study assessed whether IgE-dependent late-phase responses can be elicited in the stomach wall of mice and examined the role of mast cells in this reaction. METHODS: IgE-dependent gastric inflammation was elicited in genetically mast cell-deficient KitW/KitW-v mice, the congenic normal (+/+) mice, and mast cell-deficient KitW/KitW-v mice that had undergone local and selective reconstitution of gastric mast cell populations. RESULTS: IgE-dependent gastric reactions were associated with mast cell degranulation and the infiltration of both neutrophils and mononuclear cells in normal mice, but no significant leukocyte infiltration was observed in mast cell-deficient KitW/KitW-v mice. By contrast, in mast cell-reconstituted KitW/KitW-v mice, IgE-dependent reactions were associated with the infiltration of neutrophils and mononuclear cells. CONCLUSIONS: These results show that late-phase reactions can occur during IgE-dependent gastric inflammation in the mouse and that the infiltration of both neutrophils and mononuclear cells that are observed during this reaction are mast cell dependent.     

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals. Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea-pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site. Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils. 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7 x 10(-13) mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h. The chymase induced cell accumulation in both the guinea-pig and mouse models was dependent on an intact catalytic site, being reduced by co-injection of proteinase inhibitors or heat inactivation of the enzyme. Co-injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co-injected into the mouse peritoneum. Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.     

Impaired mast cell-dependent natural immunity in complement C3-deficient mice

The complement system is widely regarded as essential for normal inflammation, not least because of its ability to activate mast cells. However, recent studies have called into question the importance of complement in several examples of mast cell-dependent inflammatory responses. To investigate the role of complement in mast cell-dependent natural immunity, we examined the responses of complement-deficient mice to caecal ligation and puncture, a model of acute septic peritonitis that is dependent on mast cells and tumour necrosis factor-alpha (TNF-alpha). We found that C4- or C3-deficient mice were much more sensitive to caecal ligation and puncture than wild-type (WT) controls (100% versus 20% in 24-h mortality, respectively). C3-deficient mice also exhibited reductions in peritoneal mast cell degranulation, production of TNF-alpha, neutrophil infiltration and clearance of bacteria. Treating the C3-deficient mice with purified C3 protein enhanced activation of peritoneal mast cells, TNF-alpha production, neutrophil recruitment, opsonophagocytosis of bacteria and resistance to caecal ligation and puncture, confirming that the defects were complement-dependent. These results provide formal evidence that complement activation is essential for the full expression of innate immunity in this mast cell-dependent model of bacterial infection.     

Mast cell histamine-induced calcium transients in cultured human peritoneal mesothelial cells

OBJECTIVE: Peritoneal inflammation results from a complex interplay of events initiated by macrophage activity in response to infection, with the stimulation of mesothelial cell cytokine release amplifying the recruitment of blood-borne defense cells to the site of injury. Resident peritoneal mast cells may add to this complexity with mast cell derived cytokines released during this cascade. This study examined the influence of histamine, a mast cell-derived inflammatory mediator, on the initial activation of human peritoneal mesothelial cells (HPMC) by intracellular free calcium (Ca2+(i)) mobilization, and changes to the actin cytoskeleton. DESIGN: HPMC signal transduction was examined in response to histamine (1.0 mmol/L) compared to fetal bovine serum (FBS) (0.1%) and 4-br-A23187 (1.0 micromol/L). Intracellular free calcium was measured in fura-2 loaded cells with and without external calcium (Ca2+(ext)), or Ca2+(ext) with verapamil (100 micromol/L). Following treatment with agonists, HPMC actin cytoskeleton was stained using direct immunocytochemistry. RESULTS: HPMC responded to histamine with a twofold transient rise in Ca2+(i) which returned to the baseline, in contrast with FBS- and A23187-induced Ca2+(i) transients, which returned to elevated resting values. In the absence of Ca2+(ext), all agents produced a calcium transient indicative of calcium release from intracellular stores. Histamine induced calcium-dependent changes to the cytoskeleton and cellular organization, including increased actin stress fibers. CONCLUSION: Histamine produced large specific receptor-mediated calcium transients in HPMC, which included components of calcium release from intracellular stores and receptor-mediated calcium influx processes. The observed response to histamine raises the possibility that histamine derived from resident mast cells may modulate mesothelial cell function, in part by calcium-dependent pathways, and influence the performance of the peritoneal membrane during peritoneal dialysis.     

Increase in histamine synthesis by liver macrophages in CCl4-injured mast cell-deficient W/Wv mice

This study set out to examine the possible role of liver macrophages in histamine synthesis in the injured liver. The effects of the hepatotoxins Escherichia coli lipopolysaccharide (LPS) and CCl4 on histamine synthesis in the liver of mice were evaluated. C3H/HeJ mice were resistant to LPS in including histidine decarboxylase (HDC) in the liver compared with C3H/HeN mice and mast cell-deficient W/Wv mice. However, C3H/HeJ mice did respond strongly to another hepatotoxin, CCl4, leading to a significant increase in HDC activity. CCl4 also caused a marked increase in HDC activity and histamine levels in the liver of W/Wv mice. In addition, injection of CCl4 produced a large increase in the activity of HDC in the spleen and lung of W/Wv mice. HDC activity was confined to the nonparenchymal cells, with parenchymal cells expressing essentially no HDC activity. The CCl4-induced increase in HDC activity was confined, at least in part, to the liver macrophages. These results indicate that the macrophages are responsible for the increase in HDC-dependent histamine production in the liver caused by the injection of hepatotoxins. The possible role of histamine in liver regeneration after injury is discussed.     

Molecular mechanisms for the senescent cell cycle arrest

Recent advances in cutaneous mast cell biology are briefly reviewed with special reference to our own studies on cultured human mast cells. Of note are the heterogeneity of mastocytosis, the important participation of mast cells in allergic inflammation by releasing cytokines and the inhibitory effect of histamine release from mast cells by phototherapy. It is also stressed that mast cells play a major role in tissue remodelling. These novel findings suggest that mast cells can no longer be regarded simply as cells that initiate immediate allergic reactions, but that they are responsible for various chronic inflammatory or immunological events through cytokine-dependent leucocyte recruitment. The regulation of mast cell activation should be a critical issue and, thus, a promising therapeutic approach in clinical dermatology.     

Potentiation of purinergic neurotransmission in guinea pig urinary bladder by histamine

Patients suffering from the inflammatory condition of interstitial cystitis frequently exhibit an increased number of mast cells in the bladder. To determine whether mast cell mediators have the potential to influence the neurogenic contraction of the bladder smooth muscle and thereby possibly contribute to the symptoms of interstitial cystitis, we examined the effects of histamine, a major inflammatory mediator of mast cell origin, on nerve- and agonist-induced contractions of in vitro strips of guinea pig urinary bladder. Histamine (10 microM.) potentiated by more than 50% the nerve-induced contraction of bladder strips evoked by field stimulation with 0.5 msec. pulses at 4 Hz. Because the neurogenic contraction of the bladder is mediated by at least two neurotransmitters, acetylcholine (ACh) and ATP, we examined the effects of histamine on each of these transmitters. Histamine potentiated responses to the purinergic component of the neurogenic response (that part of the neurogenic response that remains after treatment with atropine) and potentiated responses to exogenously applied ATP. Histamine did not potentiate the response to the cholinergic component of the neurogenic response (that part of the neurogenic response that remains after desensitization of purinoceptors with alpha, beta-methylene ATP) nor responses to carbachol, a cholinergic agonist. These results indicate that histamine potentiates the neurogenic response of the bladder by influencing the purinergic component, apparently at postjunctional sites.     

An outbreak of measles in a highly immunised population: immunisation status and vaccine efficacy

4-Aminopyridine (4-AP) 1 mg.kg-1 sc at the scruff induced a licking response in mice. H1 receptor blockaders, such as diphenhydramine HCl (1, 10 mg.kg-1, sc at the scruff or 40 mg.kg-1, i.p.), chlorphenamine maleate (20 mg.kg-1, i.p.), and astemizole (2 mg.kg-1, i.g.), inhibited the licking response caused by 4-AP. Repeated injections of 4-AP (1 mg.kg-1) reduced the times of lick and the histamine content in the skin of injected site. 4-AP also promoted histamine release from incubated mouse peritoneal mast cells (PMC) in a dose-dependent manner. The results indicate that the licking response may originate from the histamine liberation of mast cells.     

Nutrition education in the medical school: factors critical to the development of a successful program

The phagocytosis by mononuclear phagocytes, neutrophils and eosinophils of mast cell granules which are released in the course of anaphylactic reactions was studied in the rat. Degranulation of rat peritoneal mast cells was induced either in vivo or in vitro after passive sensitization with homologous reaginic antiovalbumin serum by challenge with the antigen. The approximate extent of degranulation was assessed by determining histamine release. The anaphylactic reaction was stopped by fixation with glutaraldehyde and the cells were examined by electron microscopy. Phagocytosis was quantified in randomly selected thin section at the magnification of 1,800. Rapid and extensive phagocytosis of mast cell granules was observed both in vivo and in vitro. About one third of the mononuclear phagocytes and between 30 and 53% of the neutrophils present were engaged in phagocytosis and usually contained several mast cell granules. Phagocytosis by eosinophils was less prominent, both with respect to the proportion of phagocytosing cell (10-23%) and to the number of mast cell granules per cell profile. Examination of large numbers of cells indicates that the uptake process is highly efficient since both condensed and already disaggregated granule bodies were seen to adhere to the phagocytes and were taken up rapidly and without the need for opsonization. In the neutrophils, extensive fusion of azurophil granules (as evidenced by peroxidase cytochemistry) with phagosomes containing mast cell granules was observed. Occasionally, mast cell granules were seen within disrupted vacuoles, which could result from the swelling of the granule matrix following engulfment. The result of this study indicate that mononuclear and polymorphonuclear phagocytes have the capacity to scavenge important amounts of mast cell granule products released by anaphylaxis.     

Requirements for allergen-induced airway hyperreactivity in T and B cell-deficient mice

BACKGROUND: The pathogenesis of asthma is believed to reflect antigen-induced airway inflammation leading to the recruitment of eosinophils and activation of mast cells through cell-associated IgE. Controversies persist however, regarding the relative importance of different pathogenic cells and effector molecules. MATERIALS AND METHODS: A variety of gene-targeted mice were examined for the induction of cholinergic airway hyperresponsiveness (AH), allergic airway inflammation, mucus production, and serum IgE reactivity following intratracheal challenge with a potent allergen. AH was determined using whole-body plethysmography following acetylcholine challenge. Where possible, results were confirmed using neutralizing antibodies and cell-specific reconstitution of immune deficient mice. RESULTS: T and B cell-deficient, recombinase-activating-gene-deficient mice (RAG -/-) failed to develop significant allergic inflammation and AH following allergen challenge. Reconstitution of RAG -/- mice with CD4+ T cells alone was sufficient to restore allergen-induced AH, allergic inflammation, and goblet cell hyperplasia, but not IgE reactivity. Sensitized B cell-deficient mice also developed airway hyperreactivity and lung inflammation comparable to that of wild-type animals, confirming that antibodies were dispensable. Treatment with neutralizing anti-IL-4 antibody or sensitization of IL-4-deficient mice resulted in loss of airway hyperreactivity, whereas treatment with anti-IL-5 antibody or sensitization of IL-5-deficient mice had no effect. CONCLUSIONS: In mice, CD4+ T cells are alone sufficient to mediate many of the pathognomonic changes that occur in human asthma by a mechanism dependent upon IL-4, but independent of IL-5, IgE, or both. Clarification of the role played by CD4+ T cells is likely to stimulate important therapeutic advances in treatment of asthma.     

Gene knockout B cell-deficient mice demonstrate that B cells play an important role in the initiation of T cell responses to Chlamydia trachomatis (mouse pneumonitis) lung infection

T cell-mediated immunity as measured by delayed-type hypersensitivity, and IFN-gamma production has been shown to be critical for host defense against Chlamydia trachomatis infection in both human and animal studies. Using gene-targeted B cell-deficient mice, we examined the role of B cells in protective immunity to C. trachomatis (mouse pneumonitis) (MoPn) lung infection. B cell-deficient mice were observed to have a significantly higher mortality rate and in vivo chlamydial growth than did wild-type mice following MoPn lung infection. Interestingly, B cell-deficient mice not only lacked Ab responses but also failed to mount an efficient delayed-type hypersensitivity response following chlamydial lung infection. In contrast to results obtained from MoPn-infected wild-type C57BL/6 mice, spleen cells from infected B cell-deficient mice failed to produce Th1-related (IFN-gamma) or Th2-related (IL-6 and IL-10) cytokines after Chlamydia-specific in vitro restimulation. Moreover, unlike wild-type mice, B cell-deficient mice were not immune to rechallenge infection following recovery from primary chlamydial infection. The data indicate that B cells play an important role in host defense to primary and secondary chlamydial infection and suggest that B cells are crucial for the initiation of early T cell responses to chlamydial infection. This study provides evidence for the role of B cells in the in vivo priming of T cells during infection with the intracellular bacterial pathogen, C. trachomatis.     

Effects of thalidomide on neutrophil respiratory burst, chemotaxis, and transmigration of cytokine- and endotoxin-activated endothelium

Vascular endothelium activated by endotoxin and cytokines plays an important role in organ inflammation and blood leukocyte recruitment. Neutrophils, which are a homogeneous population of effector cells, are rapidly attracted in large numbers to sites of inflammation where they form an early response to infection or injury. Excessive production of various interleukins, TNF, arachidonic acid metabolites, and other substances by neutrophils and macrophages results in systemic endothelial cell injury, a fundamental problem. In the present study, we investigated in vitro the effects of thalidomide (THD) on activation of endothelial cells for enhanced transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), and interleukin-1 (IL-1). Modulation of endotoxin- and cytokine-induced neutrophil chemotaxis and respiratory burst by THD were also studied. Treatment of HUVEC with THD in combination with LPS, TNF, and IL-1, respectively, antagonized LPS-activated transmigration of neutrophils but stimulated the effects of TNF and IL-1. All of the agents used-THD, LPS, TNF, and IL-1-inhibited neutrophil chemotaxis. Addition of THD to the neutrophils had no effect on LPS-inhibited chemotaxis whereas the TNF- and IL-1-induced chemotaxis was modulated in a bimodal manner. However, THD failed to influence neutrophil respiratory burst activity. Results demonstrate that THD differentially affects mediator-induced activation of HUVEC and neutrophils.     

Mast cell activation is not required for induction of airway hyperresponsiveness by ozone in mice

Exposure to ambient ozone (O3) is associated with increased exacerbations of asthma. We sought to determine whether mast cell degranulation is induced by in vivo exposure to O3 in mice and whether mast cells play an essential role in the development of pulmonary pathophysiological alterations induced by O3. For this we exposed mast cell-deficient WBB6F1-kitW/kitW-v (kitW/kitW-v) mice and the congenic normal WBB6F1 (+/+) mice to air or to 1 or 3 parts/million O3 for 4 h and studied them at different intervals from 4 to 72 h later. We found evidence of O3-induced cutaneous, as well as bronchial, mast cell degranulation. Polymorphonuclear cell influx into the pulmonary parenchyma was observed after exposure to 1 part/milllion O3 only in mice that possessed mast cells. Airway hyperresponsiveness to intravenous methacholine measured in vivo under pentobarbital anesthesia was observed in both kitW/kitW-v and +/+ mice after exposure to O3. Thus, although mast cells are activated in vivo by O3 and participate in O3-induced polymorphonuclear cell infiltration into the pulmonary parenchyma, they do not participate detectably in the development of O3-induced airway hyperresponsiveness in mice.     

The mechanisms, intensity of treatment, and outcomes of hospitalized burns: issues for prevention

Our previous study revealed that passive cutaneous anaphylaxis (PCA) can be produced in congenitally mast cell-deficient WBB6F1-W/Wv (abbreviated as W/Wv) mice on sensitization with undiluted or slightly diluted allogeneic and xenogeneic antisera but not on sensitization with allogeneic monoclonal immunoglobulin (Ig)E and IgG1 antibodies regardless of the antibody concentration [1]. In view of these findings, the present study was conducted to characterize PCA in this strain from its drug susceptibilities using mast cell-bearing WBB6F1-(+)/+ (abbreviated as +/+) and B6D2F1 mice as references. PCA in W/Wv mice mediated by a low dilution (1:4) of hyperimmune serum to bovine serum albumin of the B6D2F1 mouse origin was markedly suppressed by CV-6209, an antagonist of platelet-activating factor (PAF), but not by antihistamines such as cyproheptadine and oxatomide. In contrast, PCA in +/+ and B6D2F1 mice mediated by a high dilution (1:128) of the anti-serum (virtually by IgG1 antibody) was nearly completely suppressed by antihistamines but not by CV-6209. A remarkable difference between PCA in W/Wv and reference mice was also observed in the susceptibility to monoclonal anti-mouse granulocyte (Gr-1) antibody: PCA in W/Wv mice was potently suppressed by the 1- to 3-day pretreatment with this antibody but that in references was not at all. Putting these present results together with the previous finding that anti-granulocyte antibody greatly reduces circulatory Gr-1+ leukocytes, 1 to 3 days after the treatment [2], it is highly probable that PCA in W/Wv mice mediated by some antibody isotypes other than IgE and IgG1 is produced by PAF mainly released from Gr-1+ cells, while IgG1 antibody-mediated PCA in mast cell-bearing reference mice is evoked by histamine derived from mast cells. PCA homologous to that in W/Wv mice could also be produced in the reference mice on sensitization with undiluted or slightly diluted antiserum, when generalized blueing due to excess IgG1 antibody was removed by the oxatomide treatment before the antigen challenge.     

Patterns for RANTES secretion and intercellular adhesion molecule 1 expression mediate transepithelial T cell traffic based on analyses in vitro and in vivo

Immune cell migration into and through mucosal barrier sites in general and airway sites in particular is a critical feature of immune and inflammatory responses, but the determinants of transepithelial (unlike transendothelial) immune cell traffic are poorly defined. Accordingly, we used primary culture airway epithelial cells and peripheral blood mononuclear cells to develop a cell monolayer system that allows for apical-to-basal and basal-to-apical T cell transmigration that can be monitored with quantitative immunofluorescence flow cytometry. In this system, T cell adhesion and subsequent transmigration were blocked in both directions by monoclonal antibodies (mAbs) against lymphocyte function-associated antigen 1 (LFA-1) or intercellular adhesion molecule 1 (ICAM-1) (induced by interferon gamma [IFN-gamma] treatment of epithelial cells). The total number of adherent plus transmigrated T cells was also similar in both directions, and this pattern fit with uniform presentation of ICAM-1 along the apical and basolateral cell surfaces. However, the relative number of transmigrated to adherent T cells (i.e., the efficiency of transmigration) was increased in the basal-to-apical relative to the apical-to-basal direction, so an additional mechanism was needed to mediate directional movement towards the apical surface. Screening for epithelial-derived beta-chemokines indicated that IFN-gamma treatment caused selective expression of RANTES (regulated upon activation, normal T cell expressed and secreted), and the functional significance of this finding was demonstrated by inhibition of epithelial-T cell adhesion and transepithelial migration by anti-RANTES mAbs. In addition, we found that epithelial (but not endothelial) cells preferentially secreted RANTES through the apical cell surface thereby establishing a chemical gradient for chemotaxis across the epithelium to a site where they may be retained by high levels of RANTES and apical ICAM-1. These patterns for epithelial presentation of ICAM-1 and secretion of RANTES appear preserved in airway epithelial tissue studied either ex vivo with expression induced by IFN-gamma treatment or in vivo with endogenous expression induced by inflammatory disease (i.e., asthma). Taken together, the results define how the patterns for uniform presentation of ICAM-1 along the cell surface and specific apical sorting of RANTES may serve to mediate the level and directionality of T cell traffic through epithelium (distinct from endothelium) and provide a basis for how this process is precisely coordinated to route immune cells to the mucosal surface and maintain them there under normal and stimulated conditions.