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Recently, we detected a novel biomarker in human saliva called calcium-binding protein, spermatid-associated 1 (CABS1). CABS1 protein had previously been described only in testis, and little was known of its characteristics other than it was considered a structurally disordered protein. Levels of human CABS1 (hCABS1) in saliva correlate with stress, whereas smaller sized forms of hCABS1 in saliva are associated with resilience to stress. Interestingly, hCABS1 also has an anti-inflammatory peptide sequence near its carboxyl terminus, similar to that of a rat prohormone, submandibular rat 1. We performed phylogenetic and sequence analysis of hCABS1. We found that from 72 CABS1 sequences currently annotated in the National Center for Biotechnology Information protein database, only 14 contain the anti-inflammatory domain "TxIFELL," all of which are primates. We performed structural unfoldability analysis using PONDER and FoldIndex and discovered three domains that are highly disordered. Predictions of three-dimensional structure of hCABS1 using RaptorX, IonCom, and I-TASSER software agreed with these findings. Predicted neutrophil elastase cleavage density also correlated with hCABS1 regions of high structural disorder. Ligand binding prediction identified Ca2+ , Mg2+ , Zn2+ , leucine, and thiamine pyrophosphate, a pattern observed in enzymes associated with energy metabolism and mitochondrial localization. These new observations on hCABS1 raise intriguing questions about the interconnection between the autonomic nervous system, stress, and the immune system. However, the precise molecular mechanisms involved in the complex biology of hCABS1 remain unclear. We provide a detailed in silico analysis of relevant aspects of the structure and function of hCABS1 and postulate extracellular and intracellular roles.

Rationale Sleep duration is critical to growth, learning, and immune function development in infancy. Strategies to ensure that national recommendations for sleep duration in infants are met require knowledge of perinatal factors that affect infant sleep. Objectives To investigate the mechanistic pathways linking maternal education and infant sleep. Methods An observational study was conducted on 619 infants whose mothers were enrolled at the Edmonton site of the CHILD birth cohort. Infant sleep duration at three months was assessed using the Brief Infant Sleep Questionnaire. Maternal education was collected via maternal report. Prenatal and postnatal depression scores were obtained from the 20-item Center for Epidemiologic Studies Depression Scale (CES-D). Birth records and maternal report were the source of covariate measures. Mediation analysis (PROCESS v3.0) was used to examine the indirect effects of maternal education on infant sleep duration mediated through prenatal depression and birth mode. Measurements and main results At three months of age, infants slept on average 14.1 h. Lower maternal education and prenatal depression were associated with significantly shorter infant sleep duration. Emergency cesarean section birth was associated with 1-hour shorter sleep duration at three months compared to vaginal birth [without intrapartum antibiotic prophylaxis] (β: −0.99 h; 95% CI: −1.51, −0.48). Thirty percent of the effect of lower maternal education on infant total sleep duration was mediated sequentially through prenatal depression and birth mode (Total Indirect Effects: −0.12, 95% CI: −0.22, −0.03, p Conclusions Prenatal depression and birth mode sequentially mediate the effect of maternal education on infant sleep duration.

Introduction An estimated 8.1% of Canadians adults have asthma. While there are challenges associated with the use of objective measurement of lung function in the diagnosis of asthma, we are uncertain of the barriers that impact the use of objective measures, and have limited understanding of the challenges experienced by primary care providers in diagnosis of asthma. The objectives of this quality improvement initiative were to identify primary care providers’ methods of diagnosing asthma and to identify challenges with diagnosis. Methods An online survey was disseminated using a snowball methodology. Setting Primary care practices in Alberta, Canada. Participants A total of 84 primary care providers completed the survey. Main Outcome Measures Participants were asked their ideal and sufficient methods for diagnosing asthma and to identify challenges in their practice related to asthma diagnosis. Results They identified full pulmonary function testing (54%), pre- and postbronchodilator spirometry (54%), complete history and physical (42%), peak flow measurement overtime (26%), pulmonary consult (26%), and trial of asthma medication(s) (23%), as ideal methods of diagnosing asthma. The most significant barriers to diagnosis included episodic care–care provided typically during times of worsening symptoms without ongoing preventative/maintenance care (55%), patient follow-up (44%), conflict between clinical impression and pulmonary function results (43%), patient already on asthma medications (43%), and interpreting spirometry/pulmonary function results (39%). Conclusion The results of this survey indicate that the majority of primary care providers would choose full pulmonary function testing or pre- and postbronchodilator spirometry as the ideal methods of diagnosing asthma. However, barriers related to the nature of asthma care, patient factors, and challenges with diagnostic testing create challenges. This study also highlights that primary care providers have adapted to challenges in leveraging objective measurement and may rely upon other methods for diagnosis such as trials of medications.

Background The atopic march describes the progression from atopic dermatitis during infancy to asthma and allergic rhinitis in later childhood. In a Canadian birth cohort we investigated whether concomitant allergic sensitization enhances subsequent development of these allergic diseases at age 3 years. Methods Children completed skin prick testing at age 1 year. Children were considered sensitized if they produced a wheal 2 mm or larger than that elicited by the negative control to any of 10 inhalant or food allergens. Children were also assessed for atopic dermatitis by using the diagnostic criteria of the UK Working Party. At age 3 years, children were assessed for asthma, allergic rhinitis, food allergy, and atopic dermatitis. Data from 2311 children were available. Results Atopic dermatitis without allergic sensitization was not associated with an increased risk of asthma at age 3 years after adjusting for common confounders (relative risk [RR], 0.46; 95% CI, 0.11-1.93). Conversely, atopic dermatitis with allergic sensitization increased the risk of asthma more than 7-fold (RR, 7.04; 95% CI, 4.13-11.99). Atopic dermatitis and allergic sensitization had significant interactions on both the additive (relative excess risk due to interaction, 5.06; 95% CI, 1.33-11.04) and multiplicative (ratio of RRs, 5.80; 95% CI, 1.20-27.83) scales in association with asthma risk. There was also a positive additive interaction between atopic dermatitis and allergic sensitization in their effects on food allergy risk (relative excess risk due to interaction, 15.11; 95% CI, 4.19-35.36). Conclusions Atopic dermatitis without concomitant allergic sensitization was not associated with an increased risk of asthma. In combination, atopic dermatitis and allergic sensitization had strong interactive effects on both asthma and food allergy risk at age 3 years.

Background It is hypothesised that complex interactions between genetic and environmental factors give rise to allergy and asthma in childhood. The Canadian Healthy Infant Longitudinal Development (CHILD) study was designed to explore these factors. Methods CHILD is a longitudinal, general population birth cohort study following infants from mid-pregnancy to age 5 years. Over this time period, biological samples, questionnaires, clinical measures and environmental data are collected. Results A total of 3624 families have been recruited, and many thousands of samples and questionnaires have been collected, annotated, and archived. This report outlines the rationale and methodology for collecting and storing diverse biological samples from parents and children in this study, and the mechanisms for their release for analyses. Conclusions The CHILD sample and data repository is a tremendous current and future resource and will provide a wealth of information not only informing studies of asthma and allergy, but also potentially in many other aspects of health relevant for Canadian infants and children.

It is hypothesised that complex interactions between genetic and environmental factors give rise to allergy and asthma in childhood. The Canadian Healthy Infant Longitudinal Development (CHILD) study was designed to explore these factors.CHILD is a longitudinal, general population birth cohort study following infants from mid-pregnancy to age 5 years. Over this time period, biological samples, questionnaires, clinical measures and environmental data are collected.A total of 3624 families have been recruited, and many thousands of samples and questionnaires have been collected, annotated, and archived. This report outlines the rationale and methodology for collecting and storing diverse biological samples from parents and children in this study, and the mechanisms for their release for analyses.The CHILD sample and data repository is a tremendous current and future resource and will provide a wealth of information not only informing studies of asthma and allergy, but also potentially in many other aspects of health relevant for Canadian infants and children.

Tumour necrosis factor (TNF) is an important inflammatory cytokine produced by several cell types. To test the hypothesis that there is cell-type-specific regulation and not redundancy of TNF production, we investigated its production by alveolar macrophages (AM) and peritoneal mast cells (PMC). Cell lysates of freshly isolated AM and PMC contained 9 +/- 3 pg and 57 +/- 17 pg of TNF/10(6) cells, respectively. Furthermore, unstimulated PMC expressed 4 x 10(3)-fold more attomols of TNF mRNA/microg total RNA compared with AM. These data may explain in part the greater TNF-dependent cytotoxicity of PMC. Furthermore, fixed PMC showed significantly higher TNF-dependent cytotoxic activity than AM (sevenfold), suggesting that PMC express more membrane TNF than AM. Although AM and PMC contain different amounts of TNF, antigen stimulation caused a similar release of TNF from sensitized rats. Interferon (IFN)-gamma, respectively, stimulated and inhibited AM and PMC TNF-dependent cytotoxicity whereas lipopolysaccharide (LPS) significantly stimulated TNF-dependent cytotoxicity in both cell types. However, TNF released (AM 400-fold and PMC threefold) and TNF mRNA expression, as measured by competitive reverse transcription-polymerase chain reaction (AM 7 x 10(3)-fold and PMC twofold), were considerably greater in LPS-stimulated AM than PMC. Our data indicate that TNF is differentially expressed in these two cell types and that its production is dependent on the nature of the stimulus. These data provide vital basis in experimental approaches aimed at modulating the effect of TNF in airway disease conditions involving both AM and mast cells.

Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow-derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor-alpha (TNF-alpha) storage and secretion, and TNF-alpha-dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two-dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC-associated enzyme, rat mast cell proteinase-2 (RMCP-2), but not RMCP-1, mast cell proteinase-5 (MCP-5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the periotoneal MC (PMC). BMMC demonstrated levels of TNF-alpha-dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF-alpha, in comparison with PMC, but both MC types generated substantial amounts of TNF-alpha 6 hr following IgE-mediated activation. Pretreatment of PMC with recombinant rat interferon-gamma (IFN-gamma) for 20 hr inhibited anti-immunoglobulin E (anti-IgE)-mediated release of the granule-associated enzyme, beta-hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC.

Beta 2-agonists inhibit the release of preformed mediators such as histamine and newly synthesized mediators such as prostaglandin D2 from mast cells. However, although mast cells have been identified as an important source of several cytokines including tumor necrosis factor-alpha (TNF-alpha), there is no information about their regulation by beta 2-agonists. Thus given the importance of TNF-alpha in inflammation and the widespread use of beta 2-agonists, we investigated the effect of long-acting (salmeterol) and short-acting (salbutamol) beta 2-agonists on the secretion of TNF-alpha from human skin mast cells. Treatment of mast cells with salmeterol or salbutamol (100 nmol/L) inhibited the IgE-dependent release of TNF-alpha (82% and 74%, respectively). Moreover, 2-hour treatment with salmeterol, isoproterenol, or salbutamol inhibited mast cell cytotoxicity against a TNF-alpha-sensitive cell line, WEHI-164, with an IC50 of 71, 50, and 29 nmol/L, respectively. Specificity for beta-adrenergic receptors was shown with propranolol. The inhibitory effect of beta 2-agonists was observed after only 20 minutes of treatment but was lost by 24 hours after removal of salbutamol and isoproterenol (7% and 11% inhibition remaining, respectively). In contrast, the inhibition of TNF-alpha release was increased 1 hour after removal of salmeterol and remained significant 24 hours later. Furthermore, beta 2-agonists did not show tachyphylaxis for the inhibition of TNF-alpha release. Thus selective beta2-agonists demonstrate anti-inflammatory activity by inhibiting the release of TNF-alpha from mast cells stimulated through their IgE receptor or by a tumor target cell. This inhibitory effect of beta-agonists may be important in their mode of action in the treatment of allergic diseases.

In the development of rodent mast cells (MC), IL-10 significantly enhances the growth factor activity of stem cell factor (SCF). The differential effects of IL-10 and SCF on function of rat peritoneal MC (PMC) are investigated in this study. IL-10 inhibits both constitutive and Ag-induced nitric oxide production by PMC in a dose-dependent manner, whereas SCF does not affect nitric oxide production by PMC. Short term (20-min) incubation with IL-10 does not affect Ag-induced histamine secretion, whereas long term (24-h) incubation with IL-10 significantly potentiates Ag-induced histamine secretion, an effect similar to those reported for IL-3 and IL-4 in mouse MC. By contrast, SCF significantly potentiates Ag-induced histamine secretion in both short term (20-min) and long term (24-h) experiments. Both constitutive and Ag-induced TNF-alpha production by PMC are dose-dependently inhibited by IL-10, whereas they are not affected by SCF at all doses tested (2-500 ng/ml) and incubation times observed (3-24 h). Interestingly, rat PMC constitutively express IL-10 mRNA and proteins, as tested by reverse transcription-PCR and immunocytochemistry. In addition, it was found by flow cytometry that 23.5% PMC express surface IL-10. Moreover, treatment of PMC with anti-IL-10 Ab for 6 h significantly potentiates constitutive and Ag-induced TNF-alpha production by rat PMC. Thus, IL-10 and SCF exert different regulatory effects on MC secretory function. IL-10 produced by MC has the potential to regulate MC function in an autocrine manner, an effect that may be highly relevant to responses involving MC activation.

Submandibular glands release peptides and proteins that, through exocrine and endocrine actions, facilitate tissue repair in the oral cavity, gastrointestinal tract, and more distal sites such as liver. It has been shown that salivary gland factors also modulate inflammatory responses, because we found that removal of the submandibular glands increases the hypotensive responses to endotoxin. From this observation we proposed that these glands contain a factor that regulates cardiovascular response to shock. With the use of classical peptide isolation procedures, a heptapeptide (TDIFEGG) called submandibular gland peptide T was identified in rat submandibular glands. A synthetic form of this peptide reduced endotoxic shock in sialadenectomized rats by 50% at doses as low as 1 microgram/kg and prevented allergen-induced hypotension by 90% in rats with intact salivary glands at a dose of 100 micrograms/kg. This novel peptide is probably generated from a prohormone, submandibular gland rat 1 protein, a product of the VCSA1 gene. These data indicate that submandibular glands participate in the regulation of systemic homeostasis.

The effect of stem cell factor (SCF) on histamine and tumour necrosis factor-alpha (TNF-alpha) release from rat peritoneal mast cells (PMC) was determined and the intracellular pathways involved in the potentiation of histamine secretion were investigated. The effects of SCF (2-100 ng/ml) were examined following both short-term (0 and 20 min) and long-term (up to 24hr) preincubations with SCF. Pretreatment of PMC with SCF for 0 min (concurrent) or 20 min did not induce histamine secretion directly, but significantly increased antigen (Ag)-induced histamine secretion. SCF potentiated Ag-induced intracellular Ca2+ increase and calcium ionophore A23187-induced histamine secretion. Pertussis toxin (PT) inhibited SCF-induced potentiation of IgE-dependent histamine secretion, indicating that PT-sensitive G-proteins are involved in the immediate effects of SCF. In long-term incubation experiments, SCF pretreatment for 18-24 hr significantly enhanced Ag-induced histamine secretion, but did not affect Ag-induced intracellular Ca2+ levels. The effects of long-term incubation with SCF, but not the short-term effects, were blocked by cycloheximide. Interestingly, spontaneous and Ag-induced TNF-alpha release from rat PMC were not affected by pretreatment with SCF (2-500 ng/ml) for 1 to 24 hr. Thus, through immediate and delayed mechanisms, SCF potentiates histamine release from PMC, but has not effect on TNF-alpha release. The regulation of MC by SCF may be important in allergic and other inflammatory diseases.

Sulfasalazine is an effective treatment in some inflammatory diseases that exhibit mast cell (MC) hyperplasia. However, its effect on MC has been incompletely studied. We have established that sulfasalazine inhibits the release of histamine and TNF-alpha from MC. Sulfasalazine and its metabolites, 5-aminosalicylic acid (5-ASA) and to a lesser extent sulfapyridine, inhibited Ag-stimulated histamine release from rat peritoneal MC in a concentration-dependent manner with a 50% inhibitory concentration of 6 x 10(6)M, 8 x 10(-6)M, and 3 x 10(-4)M, respectively. Similar results were observed with sulfapyridine and 5-ASA on Ag-stimulated histamine release of another population of MC, namely rat intestinal mucosal MC, but sulfasalazine was markedly less potent than its metabolites. Interestingly, sulfasalazine and sulfapyridine, but not 5-ASA, inhibited Ag-stimulated TNF-alpha released by MC. Similar results were observed with MC-mediated cytotoxic activity in which sulfasalazine and sulfapyridine, but nor 5-ASA, inhibited MC TNF-alpha-dependent cytotoxicity in a concentration-dependent manner. The addition of sulfasalazine to MC, up to 12 h after the cytotoxic assay (16 h) had started, significantly inhibited cytotoxic activity, suggesting that sulfasalazine inhibited the cytotoxic mediator, TNF-alpha. Indeed, affinity studies demonstrated that sulfasalazine binds TNF-alpha. Furthermore, the inhibition of MC cytotoxicity by sulfasalazine appeared to require new protein synthesis. Pretreatment of MC with sulfasalazine also inhibited the release of TNF-alpha and reduced the levels of TNF-alpha mRNA. Thus, sulfasalazine inhibits MC-mediated, TNF-alpha-dependent cytotoxicity by multiple mechanisms: competitive inhibition of soluble TNF-alpha, reduction of levels of TNF-alpha mRNA, and inhibition of TNF-alpha release.

SUMMARYTNF-α is a cytokine thought to be involved in the pathogenesis of asthma and in several other inflammatory conditions. Given recent evidence that mast cells (MC) are an important source of TNF-α, we investigated the effects of two anti-inflammatory drugs, nedocromil sodium (NED) and sodium cromoglycate (SCG). on rat MC-derived TNF-α. We established that at least 2h pretreatment with NED or SCG followed by washing was required to inhibit TNF-α-dependent cytotoxicity by rat peritoneal MC (PMC). A maximum inhibition of TNF-α occurred after 6h treatment. The inhibitory effect of NED and SCG (10−5 10−3 M) was concentration-dependent (20-37% for NED and 16-37% for SCG). The time-course analysis and the use of cycloheximide, an inhibitor of protein synthesis, provided strong evidence that new protein synthesis by the MC is required for this inhibitory effect. Furthermore, 24h treatment with I mM NED inhibited the levels of mRNA for TNF-α by 59-83%. In addition to the effect on TNF-α-dependent cytotoxicity by MC, 20min pretreatment with 10−4M NED and SCG inhibited antigen-stimulated TNF-α release (6h) by 42% and 48%, respectively. Interestingly, the functionally distinct intestinal mucosal MC (IMMC) is unresponsive to these drugs with regard to histamine secretion. However, as with PMC, 2h pretreatment with NED or SCG inhibited TNF-α-dependent cytotoxicity by IMMC. These effects may be important in the action of these drugs in vivo in the late phase reaction in asthma or other inflammatory conditions.

Interactions between the immune, nervous and endocrine systems are important in inflammation and tissue repair. One neuroendocrine pathway involves polypeptide factors derived from the submandibular glands, whose synthesis and release are controlled by cervical sympathetic nerves. This novel pathway of immune-neuroendocrine communication is the cervical sympathetic trunk-submandibular gland (CST-SMG) axis. Here, Ronald Mathison, Joseph Davison and Dean Befus discuss the contributions of this axis to the neuroendocrine regulation of inflammation and tissue repair.

Release of inflammatory mediators by mast cells can be modulated by certain cytokines and by nitric oxide. An in vitro platelet aggregation bioassay was used to assess the effects of interleukin-1 beta (IL-1 beta) on the release of platelet-activating factor and nitric oxide from resting or ionophore-activated peritoneal mast cells (PMC) from rat. PMC spontaneously released a substance that inhibits thrombin-stimulated platelet aggregation. The activity of this substance is abolished by addition of hemoglobin to the platelet suspension and augmented by preincubation of the PMC with L-arginine, suggesting that it is nitric oxide. Within minutes, IL-1 beta concentration-dependently (1 pg/ml-100 ng/ml) enhanced the release from activated PMC of nitric oxide, as measured by its ability to inhibit thrombin-induced platelet aggregation, and as confirmed with a biochemical assay for nitrite. This action of IL-1 beta was inhibited by pretreatment of PMC with a calmodulin antagonist (calmidazolium), an IL-1 receptor antagonist, or either of two nitric oxide synthase inhibitors (L-NAME and LY-83583). IL-1 beta also inhibited the release of platelet-activating factor from PMC through a nitric-oxide-dependent mechanism. These results demonstrate that IL-1 beta is a potent and rapid-acting modulator of mast cell reactivity, stimulating nitric oxide release while inhibiting the production of platelet-activating factor.

1. The high viscoelastic property of neutrophils is the major factor contributing to their extensive accumulation (more than 50% of circulating neutrophils) in the pulmonary microvasculature. 2. The cholinergic parasympathetic and adrenergic sympathetic nerves modulate the size of the pulmonary neutrophil pool by regulating arterial and venous pressures, increases in which promote or reduce neutrophil transit times, respectively. 3. Biochemical factors, such as the cytokines and complement, which act upon the neutrophils to increase their viscoelasticity and promote the interaction of neutrophil cell adhesion molecules with counter ligands on the endothelial cell, are the primary factors regulating the size of the pulmonary pool of vascular neutrophils. 4. The primary afferent nerves, through their release of substance P, are the most important neural elements regulating neutrophil accumulation and function. Substance P facilitates the actions of other inflammatory agents (e.g. LTB4, platelet activating factor) on neutrophil adhesion, migration and biochemical reactivity. 5. The sympathetic nervous system indirectly regulates neutrophil functions by regulating the release of an immunosuppressive factor from submandibular glands. 6. With continued, study of nervous system regulation of neutrophil function, the mechanisms by which psychological factors affect these cells will eventually be revealed.

Summary Quantitative measurements were made of different phenotypes of lymphocytes in bronchoalveolar lavage fluid (BALF) of rats during the course of a primary or secondary infection with Nippostrongylus brasiliensis. These changes were compared with those in the peripheral blood to understand the site-specificity of the responses. Following infection, there was a significant increase in both B and T lymphocytes in BALF. The CD4: CD8 ratio was significantly altered with a decreased ratio on day 2 and increased ratio on days 16 and 32 post infection (p.i.). Two colour analysis showed that during larval migration through the lungs (day 2 p.i.) there was a significant increase in CD8+, CD4+ OX22+ and CD4+ OX22- cells in BALF. As infection progressed in time, CD4+ OX22 – cells were increased significantly. Compared to primary infection, a secondary infection resulted in increased recovery of CD4+ OX22– cells in BALF. These changes were not readily appreciated in the peripheral blood, suggesting site-specific compartmentalization of lymphocyte responses in the lung. The functional significance of these dynamic changes in lymphocyte subsets in the airspaces following infection remains to be identified.

Previously we established that immediately after pretreatment with low concentrations (5-10 micrograms/ml) of formaldehyde antigen- and ionophore-induced histamine secretion was enhanced from peritoneal mast cells (PMC) isolated from rats infected with Nippostrongylus brasiliensis. In contrast to immediately following pretreatment with low concentrations of formaldehyde, 3 h after a 30-min treatment with formaldehyde (10, 50 and 100 micrograms/ml) antigen-induced histamine secretion from PMC was significantly depressed, and 35S-methionine incorporation was also decreased. To further explore the effects of formaldehyde on mast cells, we investigated protein biosynthesis of PMC following formaldehyde treatment and compared this with the effects of hydrogen peroxide (H2O2) or heat treatment. One- and two-dimensional SDS-PAGE were used to assess the effects. Formaldehyde treatment induced the synthesis of 70- and 72-kD stress-like proteins in rat PMC. Pretreatment of PMC with 50 microM H2O2 and heat (45 degrees C) also induced proteins with the same molecular weight. Two-dimensional SDS-PAGE analysis established that formaldehyde-induced 70-kD proteins had the same pI values as 70-kD heat shock proteins previously observed in mammalian cells. These results suggest that formaldehyde, H2O2 and heat shock induce stress proteins in rat PMC. It will be important to establish whether or not these stress proteins are responsible for the functional alterations observed in the mast cells.

1. Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 microM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2. This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10 microM). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20 micrograms ml-1) or a specific antibody directed against PAF. 3. The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.9 microM) or Wy-50,295 (IC50 of 1.2 microM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties. 4. Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100 microM.5. Neither PF-5901 nor Wy-50,295 (1 or 10 microM) significantly affected histamine release or prostaglandin D2 synthesis by peritoneal mast cells in response to calcium ionophore stimulation.6. These results demonstrate the ability of a class of quinoline-based compounds to inhibit PAF synthesis by peritoneal mast cells. This activity does not appear to be related to effects of these compounds on leukotriene synthesis or LTD4 receptors. The ability of these compounds to inhibit PAF synthesis may contribute to their anti-inflammatory properties.

Mast cells dispersed from human skin and purified by density-gradient centrifugation were cytotoxic toward the mouse fibrosarcoma cell line WEHI-164. Skin mast cells were not cytotoxic toward the NK cell-sensitive cell line K562. Killing of WEHI-164 occurred over a prolonged (greater than 18 h) period of incubation with mast cells and was effectively inhibited by polyclonal antibodies and mAb against TNF-alpha suggesting that this cytokine plays an important role in mast cell-mediated cytotoxicity. Whereas lysates of rat peritoneal mast cells exhibited cytotoxicity toward WEHI-164, this was not found with lysates of unstimulated skin mast cells suggesting that TNF-alpha is not stored preformed in the latter. Killing of WEHI-164 cells by skin mast cells was enhanced by anti-IgE and there was a significant correlation between histamine release and cytotoxicity after activation with this stimulus. We conclude that human skin mast cells are a potential source of TNF-alpha and suggest that these cells, particularly after activation, might contribute to the synthesis of this multifunctional cytokine in inflammatory sites.

Macrophage migration inhibitory factor (MIF) is found in immune-privileged sites and inhibits cytotoxicity mediated by CD3-ve lymphokine-activated killer cells (LAK). The mechanism by which MIF attenuates LAK cytotoxicity is unknown. We provide evidence that MIF has a major histocompatibility complex (MHC) class I-like motif. A monoclonal antibody (OX18) that binds a conserved region of rat MHC class I proteins binds native MIF. Anti-MIF polyclonal antibodies bind MHC class I. Epitope mapping suggests OX18 binds a loop of MHC class I bound by several receptors for MHC class I. A sequence (PRPEG) within the proposed OX18-binding site on MHC class I exists with a short insertion in MIF. OX18 does not bind MIF that is denatured by SDS-PAGE. This suggests the OX18 epitope is dependent on higher order structure in MIF. Interestingly, MIF inhibits binding of tetramers of MHC class I (H2D b ) to LAK cells, suggesting it may bind to receptors for MHC class I. MIF may be an example where small regions of MHC class I are used by endogenous and viral proteins to control cytotoxicity mediated by immune cells.

Although IFN enhance the cytotoxic activity of NK cells, K cells, and monocytes, IFN-alpha/beta and IFN-gamma did not stimulate the cytotoxic activity of rat peritoneal mast cells (PMC), but had an inhibitory effect. Preincubation for 2 h with 100 and 200 U/ml of IFN-gamma and IFN-alpha/beta, respectively, inhibited PMC cytotoxicity against WEHI-164 target cells. Lower concentrations of IFN-gamma (12.5 U/ml) and IFN-alpha/beta (25 U/ml) inhibited cytotoxicity of PMC after 8 h preincubation. The inhibitory effect of IFN was concentration and time dependent. In contrast to cytotoxicity, the release of histamine by PMC was not stimulated by the target cells WEHI-164 and there was no correlation between histamine release and cytotoxic activity of PMC. Specific antibody to subclasses of IFN prevented the inhibition of PMC cytotoxic activity, but preincubation with antibodies to the alternate subclass of IFN did not affect the observed inhibition. Moreover, the presence of both subclasses of IFN showed an additive inhibition of PMC cytotoxicity. The cytotoxic activity of PMC can be completely inhibited by the addition of anti-TNF during the assay. At high concentrations (400 U/ml), IFN inhibited the release of TNF from PMC. In the presence of RNA or protein synthesis inhibitors, IFN did not inhibit cytotoxicity of PMC further. We postulate that IFN may alter gene expression in mast cells in a manner that down-regulates their functions.

Four-day-old and newly excysted H. diminuta were exposed in vitro (37 degrees C; 5% CO2/air atmosphere) to serum and peritoneal cells (1.7-4.4 X 10(5] obtained from rats. Four-day-old worms incubated in serum alone were lysed in titres of less than 16. In assays containing peritoneal cells, leucocytes, predominantly eosinophils and macrophages, adhered to the posterior end of the parasite in serum titre 32, but not in serum titres 64 and 128. In this region of the worms phagocytosis of microtriches by macrophages, microthrix denudation and loss of tegument were noted. Serum-mediated lysis of newly excysted cysticercoids occurred at a serum titre of 64 and leucocyte adherence and phagocytosis of microtriches occurred in serum titres 128 and 256. Attachment of peritoneal cells to worms did not occur in assays containing heat-inactivated serum and it is suggested that regional leucocyte adherence and subsequent parasite damage is complement-mediated.

1. Changes in tissue and organ blood flow associated with sensitization of rats to the nematode parasite, Nippostrongylus brasiliensis, were studied 30 to 35 days after infection, a time when very few worms remain in the animal. 2. Neither active nor passive sensitization modified heart rate, mean arterial blood pressure, cardiac output or total peripheral resistance. Passive sensitization and administration of non-immune sera did not modify blood flow to any of the tissues studied. 3. Active sensitization increased hepatic arterial blood flow, but decreased blood flow to the stomach, duodenum, jejunum and the submandibular glands. These effects cannot be attributed to residual nematode infections as treatment with the anthelmintic, thiabendazole, did not alter blood flow relative to untreated, actively sensitized rats. 4. The effects of active sensitization on blood flow were probably due to an action of platelet-activating factor (PAF) since treatment of actively sensitized animals with the selective antagonists, WEB-2086 and BN 52021, reversed the decrease in flow seen to the intestinal regions. The PAF antagonists increased blood flow to the kidneys and the trachea of sensitized animals. 5. These results suggest that the PAF released from undetermined sources in nematode-sensitized rats, produces altered blood flow, primarily to the stomach and proximal small bowel.

Thirty days after infection with the parasite Nippostrongylus brasiliensis, microcirculatory changes associated with worm allergen-induced anaphylactic shock in pentobarbital-anesthetized rats were studied. Allergen challenge decreased heart rate, mean arterial blood pressure, and cardiac output (CO) for 60 min, although CO increased markedly by 240 min. Blood flow (ml.min-1.g wet tissue-1) was reduced to most tissues, with the exception of the kidneys, hepatic arterial, heart, and brain, for 60 min. Depressed flow to the adrenals and enhanced flow to the kidneys, trachea, stomach, and mesentery were observed at 240 min. The percent distribution of cardiac output per gram wet tissue (%CO/g) increased initially to the kidneys, heart, liver, and cervical spinal cord, decreased to the spleen and cecum, and remained unchanged to other gastrointestinal tissues. The %CO/g to most tissues returned to normal by 60 min but decreased to the adrenals at 240 min. The intestine is an important organ affected by anaphylaxis in the rat, and the liver may participate in alleviating anaphylaxis.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.

The etiology and pathogenesis of inflammatory bowel disease (IBD) is poorly understood. However, numerous studies have demonstrated that immunological and inflammatory responses are activated during this disease. A better understanding of these events will help identify appropriate therapeutic interventions. Mast cell hyperplasia is a prominent feature of inflamed intestinal tissue in IBD. Intestinal mast cells are heterogeneous and at least two populations are present in the human intestine. The authors' objective is to explore mast cell properties, activation and mediators that are involved in the induction, maintenance and perpetuation of inflammatory lesions in the intestine. Although some therapies used in IBD can modulate mast cell activities, whether these actions are important in the beneficial effects of the drugs is unknown. Future drug development targeted to the inhibition of mast cells might be of therapeutic value. However, a cascade of different cellular events are involved in IBD development. The complexity of the disease raises difficulties in the development of therapies. Multiple drugs, selective for different phases of the disease or acting on different cells, might be most appropriate, rather than a single, all-encompassing therapeutic agent.

Mast cells (MC) are important in the numerous physiological processes of homeostasis and disease. Most notably, MC are critical effectors in the development and exacerbation of allergic disorders. Nitric oxide (NO) is a diatomic radical produced by nitric oxide synthase (NOS), and has pluripotent cell signaling and cytotoxic properties. NO can influence many MC functions. Recent evidence shows the source of this NO can be from the mast cell itself. Governing the production of this endogenous NO, through alterations in the expression of tetrahydrobiopterin (BH4), a NOS cofactor, has stabilizing effects on MC degranulation. Furthermore, NO regulates the synthesis and secretion of de novo generated mediators, including leukotrienes and chemokines. These novel observations add to the growing body of knowledge surrounding the role of NO in the MC.

To evaluate the effectiveness of a comprehensive asthma management education program for 7- to 12-year-old children with asthma, entitled Roaring Adventures of Puff (RAP), 18 elementary schools in Edmonton were randomized to intervention and control groups. Participating in the program were 76 students with asthma in the intervention schools and 86 in the control schools. Children in the intervention schools had statistically significant improvements in unscheduled doctor visits, missed school days, moderate-to-severe parent rating of severity, severity of shortness of breath, limitations in the kind of play, and correct use of medications. Unscheduled doctor visits and missed school days were the only significant improvements in the control group; however, improvements were about half that of the intervention group. The results showed that a comprehensive, school-based asthma education program is feasible and improves outcomes.

In this work we analyzed by RT-PCR, the mRNA changes for IL-4, IL-10, TNF and IFN (induced by TSL-1 antigens in a rat mast cell line (HRMC) with mucosal characteristics. The data obtained showed an increase of 65 and 52% in mRNA expression for IL-4 and TNF respectively and a decrease of 59 and 55% in mRNAs for IFN gamma and IL-10. Our results suggest that TSL-1 antigens induce the release from MC of regulatory molecules, such as IL-4 by an IgE independent mechanism. Our data also provides important information related to the ability of MC to participate not only in the effector phase against the infectious agents, but also in the orchestration of the immune response by the host against parasites.

Neuro-endocrine immunology, a field arising from curiosity about the mind-body connection, is evolving rapidly. From intriguing, but seemingly unexplainable observations with human infections and disease, experimental systems have been developed that provide a solid scientific basis for new understanding. There have been major efforts to understand influences of the nervous system on immune and inflammatory responses, e.g., innervation of the immune system, molecular communication pathways, and complex phenomena such as conditioning of immune responses and mechanisms of host defenses. In turn, the immune system communicates with the neuro-endocrine systems. Imbalances in the neuro-endocrine-immunologic circuitry are relevant in host defenses and in injury and repair. Examples of these themes in neuro-endocrine-immunology arise in several host-parasite models of neurogenic inflammation, immediate hypersensitivity responses, and granuloma formation. The hypothalamic-pituitary-adrenal axis and the cervical sympathetic trunk-submandibular gland axis provide important models to enhance understanding of this poorly known component of the host-parasite relationship.

The "Roaring Adventures of Puff" (RAP) is a childhood asthma education program that targets children 7 to 12 years of age and their families. This program is designed to improve the quality of life of these children and improve asthma management for optimal asthma control. The program uses interactive educational strategies based on the principles of the Social Cognitive Theory. The article highlights aspects of the theory, provides an overview of the RAP program, and outlines some of the practical components of the program. RAP is an innovative program that is adaptable to a variety of settings and may serve as a health education model for children.

CD8, a marker largely restricted to subsets of T lymphocytes and NK cells, was detected on freshly isolated rat peritoneal mast cells (PMC). Using flow cytometry, Percoll-enriched rat PMC (or = 98% purity) were positive for the hinge region of CD8alpha (67.5 +/- 9.5%; Ab OX8) and CD8beta (27.8 +/- 2.3%; Ab 341). CD8+ PMC consisted of two populations, CD8alpha+ (22.5%) and CD8alpha+ beta+ (15.9%). Interestingly, G28, an Ab that identifies the IgV-like region of CD8alpha on T lymphocytes, did not bind PMC, suggesting that PMC CD8alpha is distinct from that on T lymphocytes. Moreover, a similar pattern of Ab positivity for CD8 was observed on a rat mast cell line, RBL 2H3. The presence of CD8alpha immunoreactivity on rat PMC was further confirmed by confocal microscopy. In situ reverse-transcription PCR and reverse-transcription PCR analysis demonstrated that PMC contained mRNA transcripts encoding CD8alpha. In functional studies of CD8 on PMC, both TNF-alpha and nitric oxide production were induced by OX8 (CD8alpha) and 341 Ab (CD8beta) in a dose-dependent manner. However, neither OX8 nor 341 induced histamine secretion from PMC. Ag-induced secretion of TNF-alpha, nitric oxide, and histamine was not affected by OX8 or 341 Abs, suggesting that there are distinct signaling mechanisms mediated by CD8 and Fc epsilonRI. These results indicate that rat PMC express functional CD8 molecules that may be distinct from those of T lymphocytes. The difference suggests there is a ligand other than MHC class I for mast cell CD8.

Prior studies demonstrated that rat macrophages express CD8, which differs from T lymphocyte CD8 within the ligand binding domain. We investigated whether stimulation of macrophage CD8 could induce mediator release and regulate host defense. Cross-linking either CD8alpha (OX8, 5 microg/ml) or CD8beta (341, 10 microg/ml) stimulated nitric oxide (NO) production, which correlated with an up-regulation of inducible NO synthase protein. Cell signaling inhibitors were used to elucidate the pathways of CD8alpha and CD8beta stimulation. Genistein (broad spectrum protein tyrosine kinase inhibitor, 10 microg/ml), PP1 (src family kinase inhibitor, 5 microg/ml), polymyxin B (protein kinase C (PKC) inhibitor, 100 microg/ml), and Ro 31-8220 (PKC inhibitor, 1 microM) significantly inhibited anti-CD8alpha- and anti-CD8beta-stimulated NO production and inducible NO synthase up-regulation, suggesting that tyrosine kinase(s) (src family) and PKC are involved in CD8 signaling. In addition, cross-linking CD8alpha stimulated NO-dependent macrophage killing of the parasite Leishmania major. For the first time, this work demonstrates that the beta-chain of macrophage CD8, in addition to the alpha-chain, can regulate mediator release. These results further illustrate the importance of this molecule and support our previous data demonstrating differences between macrophage and T lymphocyte CD8. Additional studies on the signaling mechanisms and possible ligand(s) for macrophage CD8 will lead to a greater understanding of inflammation and host defense.

Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile -induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-α). In contrast, 1 μg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 μg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response to C. difficile infection by releasing TNF-α upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficile toxins.

Beta 2-agonists inhibit the release of preformed mediators such as histamine and newly synthesized mediators such as prostaglandin D2 from mast cells. However, although mast cells have been identified as an important source of several cytokines including tumor necrosis factor-alpha (TNF-alpha), there is no information about their regulation by beta 2-agonists. Thus given the importance of TNF-alpha in inflammation and the widespread use of beta 2-agonists, we investigated the effect of long-acting (salmeterol) and short-acting (salbutamol) beta 2-agonists on the secretion of TNF-alpha from human skin mast cells. Treatment of mast cells with salmeterol or salbutamol (100 nmol/L) inhibited the IgE-dependent release of TNF-alpha (82% and 74%, respectively). Moreover, 2-hour treatment with salmeterol, isoproterenol, or salbutamol inhibited mast cell cytotoxicity against a TNF-alpha-sensitive cell line, WEHI-164, with an IC50 of 71, 50, and 29 nmol/L, respectively. Specificity for beta-adrenergic receptors was shown with propranolol. The inhibitory effect of beta 2-agonists was observed after only 20 minutes of treatment but was lost by 24 hours after removal of salbutamol and isoproterenol (7% and 11% inhibition remaining, respectively). In contrast, the inhibition of TNF-alpha release was increased 1 hour after removal of salmeterol and remained significant 24 hours later. Furthermore, beta 2-agonists did not show tachyphylaxis for the inhibition of TNF-alpha release. Thus selective beta2-agonists demonstrate anti-inflammatory activity by inhibiting the release of TNF-alpha from mast cells stimulated through their IgE receptor or by a tumor target cell. This inhibitory effect of beta-agonists may be important in their mode of action in the treatment of allergic diseases.

A novel peptide, submandibular gland peptide-T (SGP-T), which reduces allergen-induced hypotension, was examined for effects on intestinal anaphylaxis. Hooded-Lister rats were sensitized to egg albumin and prepared for the measurement of in vivo myoelectric activity of the jejunum. The disruption of migrating myoelectric complexes (MMCs) that occurs upon intraluminal, duodenal challenge with antigen of sensitized rats was inhibited by 75% upon intravenous treatment with 100 micrograms/kg of SGP-T. In addition, SGP-T reduced the number of rats experiencing anaphylactic diarrhea and disrupted MMCs, but the peptide did not alter antigen-provoked release of rat mast cell protease II. The mechanism of action of SGP-T remains to be determined, but it apparently does not act directly on mast cells to exert its antianaphylactic action. These results emphasize that modulation of immediate hypersensitivity reactions is only one of several gastrointestinal activities that are affected by growth factors and peptides released from salivary glands.

Macrophages play an essential role in host defense, and we have identified a novel CD8 molecule, on alveolar and peritoneal macrophages, that may be involved in regulating this function. Flow cytometric analysis of bronchoalveolar lavage from normal rats identified a large number of CD8-positive cells that could not be accounted for by T lymphocytes. Within the scatter profile region in which the majority of cells were alveolar macrophages (OX41; 89 +/- 1%), 63 +/- 5% of the cells stained positively for CD8alpha (OX8) and 52 +/- 3% for CD8beta (341). Double-staining of lavage cells confirmed the presence of CD8 on alveolar macrophages. Interestingly, flow cytometry showed differences between CD8 on alveolar macrophages and on T lymphocytes within the ligand binding domain for MHC class I. Reverse transcription-PCR analysis on FACS-enriched alveolar macrophages showed the presence of CD8alpha mRNA, determining that macrophages synthesize CD8. Further studies identified both the alpha (49 +/- 8%)- and beta (37 +/- 4%)-chains of CD8 on peritoneal lavage cells (86 +/- 3% macrophages (OX42, CD11b)). As with alveolar macrophages, there were differences within the ligand-binding domain of CD8 on peritoneal macrophages compared with T lymphocytes. Functional studies determined that anti-CD8alpha (OX8) stimulated a dose-dependent release of nitric oxide, indicating that CD8 can directly regulate macrophage function. Thus, macrophages express an unusual CD8 molecule that differs within its ligand-binding domain, compared with T lymphocytes, and these findings suggest hitherto unknown ligand(s) for CD8. These findings will lead to a greater understanding of macrophage function and regulation.

Rat mast cell lines (hybrid rat mast cells, HRMC, and rat cultured mast cells, RCMC) and mast cells from the rat body cavity were used to test the hypothesis that IFN-alpha/beta and IFN-gamma inhibit tumor necrosis factor alpha (TNF-alpha)-mediated cytotoxicity by depressing the steady-state levels of mRNA for TNF-alpha. In vitro treatment of mast cells with IFN-gamma and IFN-alpha/beta depressed mRNA levels. By contrast, IFN pretreatment of mast cell lines induced an increase in levels of mRNA for the IFN-inducible gene, 2'5'-oligoadenylate synthetase and also for high-affinity IgE-receptor-alpha. The IFN-mediated regulation of mast cells may be an important mechanism in the control of inflammatory pathways characterized by Th1- and Th2-type responses.

Six basic proteins of 26 to 38 kDa with isoelectric points (pI)or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pIor = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.

We have further characterized the heterogeneity of mast cells (MCs) by comparing the ability of rat peritoneal MCs (PMCs) and intestinal mucosal MCs (IMMCs) to produce tumor necrosis factor (TNF)-alpha and by investigating its regulation by interferon (IFN) and the antiallergic drugs nedocromil sodium (NED) and sodium cromoglycate (SCG). Although IMMCs store less TNF-alpha than PMCs, they produced comparable amounts of TNF-alpha in cytotoxic assays. Just as SCG and NED inhibit histamine secretion from PMCs but not IMMCs, IFN exhibited a similar differential effect on histamine release from these cells. However, SCG, NED, and IFN inhibit TNF-alpha-dependent cytotoxicity by both PMCs and IMMCs and reduce the steady-state levels of mRNA for TNF-alpha in PMCs. Thus, the modulation of MC mediator release depends upon the MC population and mediator studied. The inhibitory effect of SCG and NED on TNF-alpha release from MCs may explain some of their anti-inflammatory and therapeutic effects.