Modulation of cellular immune function in vitro by histamine receptor-bearing lymphocytes: mechanism of action.

When soluble histamine is added to guinea pig lymphocytes in vitro, antigen-induced cellular proliferation and the production of migration inhibitory factor is suppressed. The inhibitory effects that are produced by histamine have been shown to be mediated by the histamine-type 2 receptors of the involved cells, but the exact nature of this suppression has not been fully explored. The present studies have evaluated, following immunization, the effect of histamine on macrophage function in vitro, and affinity chromatography to delete a subpopulation of cells bearing histamine receptors. When we treated monolayers of peritoneal exudate cells with histamine (up to 10^?3M) we found that histamine did not interfere with antigen binding by macrophages, macro phage presentation of antigen to lymphocytes, nor the antigen-independent or antigen-dependent lymphocyte-macrophage rosetting. Columns containing insolubilized conjugates of histamine and rabbit serum albumin depleted a subpopulation of cells responsive to histamine i.e., the non-adherent cells made migration inhibitory factor and proliferated in the presence of histamine. The latter finding suggested that the retained cells might have suppressor function and if so, might mediate their effect through the release of a soluble factor. Preliminary data obtained in these studies supports this hypothesis. We conclude that cells bearing histamine receptors may serve a regulatory role in cellular immunity after their activation by histamine by producing a non-dialyzable factor with immunosuppressive properties.     

Histamine stimulates human lung fibroblast migration

Histamine is a potent mediator in allergic inflammatory processes and is released by basophils and mast cells. The aim of this study was to investigate the effect of histamine on in vitro migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn), a chemoattractant. Using the blindwell chamber technique, histamine alone had no chemotactic activity. However, histamine augmented HFn-induced HFL-1 migration at concentrations ranging between 0 and 10^?7 M (290.6 ± 20.8%) (P < 0.05). The concentration-response was bell-shaped. The effect of histamine increased with time. The stimulatory effect of histamine on HFL-1 migration was inhibited by an H4 receptor antagonist, JNJ7777120 (10^?5 M). Histamine’s effect was also inhibited by pertussis toxin (50 ng/ml), showing that the effect was mediated by the H4 receptor. This study demonstrated that histamine has the potential to stimulate human lung fibroblast migration, and thus may contribute to regulation of wound healing and the development of fibrotic disorders of the lung.     

Functional characteristics of histamine receptor-bearing mononuclear cells. II. Identification and characterization of two histamine-induced human lymphokines that inhibit lymphocyte migration

Although functional histamine receptors have generally been restricted to those human T lymphocytes expressing suppressor cell functions, more recent evidence suggests that histamine receptor-bearing human T lymphocytes are functionally heterogeneous and capable of other immunomodulatory activities. Lymphocyte chemoattractant factor (LCF) is a cationic sialoprotein with an apparent m.w. of 56,000, whose production is limited to histamine-type 2 receptor-bearing human T cells. LCF is selectively chemokinetic for T lymphocytes, and presumably contributes to the recruitment of unsensitized effector lymphocytes at inflammatory sites. In addition to LCF, Sephadex G-100 gel filtration of histamine-induced lymphocyte supernatants revealed two regions of migration inhibitory activity for human blood T and rat splenic lymphocytes. These regions corresponded to m.w. of 70,000 to 80,000 (LyMIF75K) and 30,000 to 40,000 (LyMIF35K). LyMIF75K had a single pI of 7.5 to 8.0, and its biologic activity was sensitive to trypsin but not to neuraminidase or heat (56 degrees C). LyMIF35K had a single pI of 8.5 to 8.8, and its biologic activity was sensitive to neuraminidase and heat but not to trypsin. These LyMIFs therefore appeared to be distinct from one another and physicochemically different from other migration inhibitory lymphokines. All three lymphokine activities appeared within 4 hr of incubation. The minimum concentration of histamine required to stimulate production of the LyMIF was 10(-6) M. Lymphocytes that did not adhere to a histamine affinity matrix were unable to produce either LyMIF upon subsequent stimulation with histamine or concanavalin A (Con A). Lymphocytes incubated with histamine and diphenhydramine produced LCF but neither LyMIF, whereas cells incubated with histamine in the presence of cimetidine produced both LyMIF but not LCF. These data suggest that a subset of lymphocytes defined by the presence of histamine-type 1 receptors are capable of producing two distinct species of lymphocyte migration inhibitory activity. These cells may contribute to the immobilization of effector T lymphocytes chemokinetically attracted to certain inflammatory sites.     

Histamine-induced suppressor factor (HSF): further studies on the nature of the stimulus and the cell which produces it.

Guinea pig lymphocytes are stimulated by histamine to produce a soluble factor with immunosuppressive properties. This factor, termed histamine-induced suppressor factor or HSF, abrogates the production of migration inhibitory factor (MIF) and proliferative response to specific antigen. In the present study we have determined the lymphocyte subpopulation which elaborates HSF, the lymphoid tissue source, the kinetics of its generation in relation to immunization, and the nature of the histamine receptor involved in modification of the release of HSF. HSF activity could be detected in populations of cells from spleen and lymph nodes prior to active immunization of the donor, but not in cells from the donor's blood or thymus. Following immunization with ortho-chloro benzoyl-bovine γ-globulin in complete Freund's adjuvant (CFA), more HSF activity was detected in cells from the donor's spleen and lymph nodes. The peak response was seen 2 weeks postimmunization when significant amounts of HSF also were made by cells from the blood and thymus. Concentrations of T-cell-enriched and B-cell-enriched populations were tested for their ability to make HSF. We found that T-cell-enriched, but not B-cell-enriched populations, made significant amounts of HSF. Cells from the lymph nodes of immunized donors were chromatographed over affinity columns made of insolubilized conjugates of histamine with albumin. The nonretained cells were unable to generate HSF, whereas HSF activity was detected in the cells that were retained by the columns. This finding strongly suggests that the HSF-producing cells have receptors for histamine. Cells from CFA-immune lymph nodes were incubated with H₁ (2-methyl histamine) and H₂ (4-methyl histamine) agonists to determine their relative potency and, therefore, the nature of the histamine receptors on these cells that were modifying HSF release. Although both agonists could induce generation of HSF when high concentrations (10^?3M) were used, only the H₂ agonist stimulated production or release of HSF at lower concentrations (10^?5M). These HSF-producing cells appear to be selectively sensitive to H₂ agonists and likely have a predominance of H₂ receptors. Allergic mediators other than histamine were studied to determine their ability to allow elaboration of HSF-like activity from CFA-immune lymph node cells. Serotonin (10^?3M), slow-reacting substance of anaphylaxis (100 units/ml), eosinophil chemotactic factor (tetrapeptide; 10^?5M), and prostaglandin E₁ (10^?4M) were unable to induce HSF-like activity in lymph node cells from donors immunized with CFA. Furthermore, other agents which raise intracellular levels of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) such as isoproterenol and cholera toxin, as well as the dibutyryl form of cyclic AMP itself, were also unable to generate HSF-like activity. Thus, histamine is unique among the allergic mediators in stimulating elaboration of the suppressive substance. These findings also suggest that the ability of histamine to stimulate HSF may not reside in the conventional pathway linked to cAMP accumulation, but rather to an as yet undefined pathway of cell activation. A model is presented which further implicates histamine as a modulator of cellular immune reactions.     

Complement-mediated release of histamine from human leukocytes.

Activation of either the alternative or classical pathway of complement generated a factor which induced release of histamine from both non-allergic and allergic human basophils. This factor probably is derived from the complement system since 1) its formation was associated with loss of C3 activity in human serum, 2) chemotactic factor, probably also a complement product, was generated simultaneously, 3) heat inactivation blocked its formation, 4) anti-C3 and anti-C5 blocked formation of the factor, and finally 5) anti-C5 inhibited the activity of the factor once it had been formed. It appears that both complement-mediated and allergen-mediated release of histamine from basophils are secretory, non-cytolytic pathways since both were maximal at 37 degrees C, required the presence of divalent cations, and were inhibited by theophylline. One consistent difference between these two mechanisms was noted: complement-initiated release of histamine occurred more quickly.     

Identification of a proton-chloride antiporter (EriC) by Himar1 transposon mutagenesis in Lactobacillus reuteri and its role in histamine production

The gut microbiome may modulate intestinal immunity by luminal conversion of dietary amino acids to biologically active signals. The model probiotic organism Lactobacillus reuteri ATCC PTA 6475 is indigenous to the human microbiome, and converts the amino acid l-histidine to the biogenic amine, histamine. Histamine suppresses tumor necrosis factor (TNF) production by human myeloid cells and is a product of l-histidine decarboxylation, which is a proton-facilitated reaction. A transposon mutagenesis strategy was developed based on a single-plasmid nisin-inducible Himar1 transposase/transposon delivery system for L. reuteri. A highly conserved proton-chloride antiporter gene (eriC), a gene widely present in the gut microbiome was discovered by Himar1 transposon (Tn)-mutagenesis presented in this study. Genetic inactivation of eriC by transposon insertion and genetic recombineering resulted in reduced ability of L. reuteri to inhibit TNF production by activated human myeloid cells, diminished histamine production by the bacteria and downregulated expression of histidine decarboxylase cluster genes compared to those of WT 6475. EriC belongs to a large family of ion transporters that includes chloride channels and proton-chloride antiporters and may facilitate the availability of protons for the decarboxylation reaction, resulting in histamine production by L. reuteri. This report leverages the tools of bacterial genetics for probiotic gene discovery. The findings highlight the widely conserved nature of ion transporters in bacteria and how ion transporters are coupled with amino acid decarboxylation and contribute to microbiome-mediated immunomodulation.     

Histamine modulation of eosinophil migration.

Preincubation of eosinophils with 10(-5) M or higher concentrations of histamine inhibited the eosinophil chemotactic response to endotoxin-activated serum whether by using the nucleopore filter assay and counting the cells migrating through the filter, or by using the Zigmond-Hirsch assay and counting the cells at each 10-mum interval. When the H2-receptor sites on the eosinophils were blocked by metiamide, the inhibitory capacity of histamine was prevented. Preincubation of eosinophils with 10(-6) M histamine increased the number of responding eosinophils to endotoxin-activated serum and this enhancement was blocked by an H1-receptor antagonist. Isoproteronol and aminophylline inhibited eosinophil movement and increasing concentrations of dibutryl cyclic AMP inhibited eosinophil migration. Concentrations of histamine that consistently resulted in inhibition of eosinophil movement stimulated an increase in cyclic AMP that was prevented by blocking the H2-receptor but not the H1-receptor. Thus, histamine-dependent inhibition of the eosinophil chemotactic response to other agents is mediated through the H2-receptor and is associated with an increase in the intracellular level of cyclic AMP whereas histamine dependent enhancement of eosinophil migration to other agents appears to be mediated through the H1-receptor. Eosinophils behave as a heterogeneous population as assessed by the ability of histamine to augment or inhibit cell migration. This may reflect differences in H1 to H2 receptor density or cell responsiveness to receptor stimulation. The chemoattractant activity of histamine itself is not influenced by H1 or H2 receptor antagonists, thus it is possible that an eosinophil has a third type of histamine receptor.     

Feedback suppression of the immune response in vivo. I. Immune B cells induce antigen-specific suppressor T cells

Stimulated lymphocytes are capable of synthesizing and secreting a variety of lymphokines which can affect the functions of several types of target cells. We report here the existence of a soluble factor released by activated human mononuclear leukocytes which produces a selective inhibition of human pulmonary fibroblast migration. This fibroblast migration inhibitory factor (FIF) was produced by antigen- or mitogen-stimulated human peripheral blood mononuclear leukocytes (PBML) and purified T cells. It inhibited the migration of ⁵¹Cr-labeled fibroblasts in a dose-dependent fashion with optimal effect (65–70% inhibition) obtained at 1:10 dilution and 8–20 hr of incubation. Sephadex G-100 fractionation revealed most activity to be found between 28,000 and 34,000 daltons. FIF was stable at 56 °C for 15 min, but destroyed at 80 °C or at low pH. This factor may play an important role in the modulation of fibrogenesis and healing processes by the immune system.     

Characteristics and mechanisms of action of the concanavalin A-activated suppressor cell in man.

Human peripheral blood lymphocytes treated for 24 to 48 hr with optimally mitogenic doses of concanavalin A suppressed the proliferative response of autologous T cells to mitogens and antigens. Con A-treated cells also suppressed the proliferative response and the immunoglobulin synthetic response of autologous B cells stimulated in vitro by T cell helper factor. The human Con A suppressor cell was sensitive to treatment with mitomycin C and to exposure to radiation doses exceeding 1000 rads. The Con A suppressor cell was shown to reside in the nylon wool-nonadherent, sheep red cell rosette-forming, histamine receptor-bearing population of lymphocytes and to lack surface DRW antigens. One mechanism of action of Con A suppressor cells was shown to be the inactivation of nonspecific T cell helper factor.     

Complement-induced histamine release from human basophils. I. Generation of activity in human serum.

The incubation of zymosan, endotoxin, or immune aggregates with normal human serum activates a factor which induces release of histamine from autologous basophils. The reaction can be divided into two steps: in the first, complement must be activated and in the second, the histamine-releasing factor interacts with basophils. The generation of histamine-releasing activity in serum occurs at 17 to 37 degrees C but not at 0 degrees C, is inhibited by heating the serum at 56 degrees C for 30 min, or by the addition of EDTA to the serum. Once generated, the histamine-liberating activity is stable to heating at 56 degrees C for 30 min. Gel filtration of the activated serum demonstrated that this factor eluted in the same region as a factor with chemotactic activity. Both factors have a molecular weight of about 16,000 daltons and their activities were inhibited by antibody to human C5. This is therefore a pathway for histamine release by C5a where the activation of the basophil is unrelated to the membrane bound IgE.     

The effect of pKa on pyrimidine/pyridine-derived histamine H4 ligands

During the course of our efforts toward the discovery of human histamine H₄ antagonists from a series of 2-aminiopyrimidines, it was noted that a 6-trifluoromethyl group dramatically reduced affinity of the series toward the histamine H₄ receptor. This observation was further investigated by synthesizing a series of ligands that varied in pK_a of the pyrimidine derived H₄ ligands by over five orders of magnitude and the effect on histamine H₄ affinity. This trend was then extended to the discovery of C-linked piperidinyl-2-amino pyridines as histamine H₄ receptor antagonists.     

Generation of, lipid neutrophil chemoattractant activity by histamine-stimulated cultured endothelial cells

Endothelial cell-neutrophil interactions are an important aspect of inflammatory responses. Because vascular endothelial cells respond to the inflammatory mediator histamine, these studies determined whether histamine could induce endothelial cells to release substances that affect human neutrophil migration. Cultured bovine and human endothelial cells incubated with histamine released neutrophil chemoattractant activity within 1 min; peak levels were noted in 45 min. Cimetidine, an H2 receptor antagonist, blocked chemoattractant production, whereas diphenhydramine, an H1 receptor antagonist, did not. Cycloheximide did not inhibit release of chemoattractant activity, suggesting de novo protein synthesis was not necessary for its appearance. Extraction with acidified diethyl ether partitioned all neutrophil chemoattractant activity into the organic phase. The lipoxygenase pathway inhibitors, diethylcarbamazine and 5,8,11,14 eicosatetraynoic acid, inhibited generation of this lipophilic chemoattractant activity, whereas indomethacin, a cyclo-oxygenase inhibitor, did not. Resolution of the histamine-induced endothelial cell-derived chemoattractant activity by reverse-phase high pressure liquid chromatography yielded several peaks of chemoattractant activity, none of which co-eluted with leukotriene B4, platelet-activating factor, or two mono-hydroxyeicostetraenoic acids. These findings suggest that endothelial cells release lipid neutrophil chemoattractant activity that may play a role in inflammatory responses associated with histamine.     

Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.     

Production of neutrophil-specific lipid chemoattractant activity by cultured endothelial cells: heterogeneity dependent on species, ligand, or endothelial cell site of origin.

Previous studies have demonstrated that the interaction of cultured bovine aortic and pulmonary arterial endothelial cells and the proinflammatory vasoactive amines histamine, serotonin, and angiotensin II, causes production of three novel lipid neutrophil-specific chemoattractants that are distinct from other phospholipid or lipid neutrophil chemoattractants. In this study, we investigated the species and site specificity of this inflammatory response by incubating human aortic and pulmonary arterial endothelial cells with histamine, serotonin, and angiotensin II and assaying the supernatants for their effect on neutrophil migration. Each of these vasoactive amines caused production of neutrophil chemoattractant activity in a concentration dependent manner in both cell types. For each amine, production was blocked by a specific antagonist: cimetidine for histamine, methiothepin for serotonin-stimulated aortas, ketanserin for serotonin-stimulated pulmonary arteries, and saralasin for angiotensin II. In each case, all chemoattractant activity partitioned into the organic phase and resolution by HPLC yielded two chemotactic lipids. As with the lipid chemoattractants produced by bovine endothelial cells, these lipids did not coelute with PAF, LTB4, 5-HETE, or 15-HETE, nor did they increase lymphocyte or monocyte migration. The pattern of chemotactic activity following resolution by HPLC was similar in both human aortic and pulmonary arterial endothelial cells, but was different from that of bovine aortic and pulmonary arterial endothelial cells in that only two chemoattractant lipids appeared; the third chemotactic lipid was never produced. These studies demonstrate that human endothelial cells may actively participate in neutrophil enriched local inflammatory responses by production of neutrophil-specific chemotactic factors. They also suggest this response may be dissimilar depending on the site and species from which the endothelial cells originate.     

A nonspecific inhibitor of leukocyte migration in serum from rats bearing large fibrosarcomata.

A 3 M KCl crude extract of the syngeneic benzpyrene-induced fibrosarcoma termed BP 238 specifically inhibits migration out of glass capillary tubes of immune spleen cells from tumor amputee and small tumor-bearing rats, as does supernatant medium from tumor cells grown in culture. Serum from rats bearing small (< 2 cm³) tumors does not inhibit migration of immune spleen cells, while serum from rats with larger tumors (>4 cm³) nonspecifically inhibits migration of both immune and nonimmune spleen cells, thoracic duct lymphocytes, and thymocytes. This nonspecific inhibition increases with increasing tumor size, does not correlate with the presence of bacterial infection, and is presumably due to a circulating factor produced in vivo during tumor growth. Production of macrophage inhibitory-like factor (MILF) by neoplasms in vivo may offer a mechanism for tumors to escape immunosurveillance by systemic immobilization of cytotoxic lymphocytes. From Sephadex and ultrafiltration fractionation experiments, the molecular weight of MILF in serum is polydisperse (30,000–100,000 daltons), and is heat and chymotrypsin resistant, in contrast with the properties reported for LIF (leukocyte inhibitory factor) and MIF (macrophage migration inhibitory factor) produced in vitro.     

Initiation and orientation of the symbiote migration in the human body louse Pediculus humanus L.

Bacterial symbiotes in the human body louse Pediculus humanus migrate from the mycetome to the lateral oviducts during the adult moult. Experimental results are presented suggesting that the symbiote migration is initiated by a humoral factor associated with the adult moult; that the factor is present in both sexes but the male mycetome is unable to respond to it; and that the symbiotes are attracted by the female reproductive tract.     

IV. Immunologic mechanisms for release of chemical mediators of anaphylaxis from human lung tissue

The antigen-induced, IgE-dependent release of chemical mediators from human lung tissue in vitro is modulated by a variety of pharmacologic maneuvers involving alterations in the intracellular levels of cyclic nucleotides. Increase in the level of cyclic AMP inhibits the immunologic release of histamine, SRS-A and ECF-A; β-adrenergic agents, prostaglandins, cholera toxin and methylxanthines all produce accumulations of cAMP in human lung tissue. Depletion of cAMP after α-adrenergic, low-dose prostaglandin and imidazole stimulation is associated with enhancement of the release of mediators. Studies involving purified preparations of rat peritoneal mast cells confirm that alterations in the cAMP levels of a homogeneous population of target cells indeed influence histamine release in a fashion analogous to that of human lung tissue.Furthermore, cholinergic stimuli produce a marked enhancement of the antigen-induced release of mediators from human lung through an apparently independent mechanism, presumably acting through alterations in the tissue concentration of cyclic GMP. This latter observation suggests an important interaction between endogenously released parasympathetic neurohormones and the immunologic release of the chemical mediators of asthma.     

Histamine-induced suppressor factor (HSF): effect on migration inhibitory factor (MIF) production and proliferation.

Histamine added in vitro to cultures of sensitized lymphocytes suppresses antigen-induced production of migration inhibitory factor (MIF) and proliferation by these cells. Recent studies have suggested that lymphocytes bearing histamine type-2 receptors play a regulatory role in these in vitro responses. The present studies were undertaken to determine if suppressor function by cells having histamine receptors was mediated through a soluble product. It was found that lymph node cells from nonimmune or immune strain 2 guinea pigs elaborate a nondialyzable factor into the culture supernatant when incubated with 10(-3) to 10(-5) M histamine (histamine-induced suppressor factor of HSF). HSF, when cocultured with sensitized lymphocytes, suppressed their MIF and proliferative responses to antigen. HSF was made by lymphocytes but not macrophages. Its production could be blocked by an H2 receptor antagonist (burimamide) but not an H1 receptor antagonist (chlorpheniramine). Furthermore, the inhibitory effect of HSF was reversible as lymphocytes washed free of the factor after 24 hr and recultured with fresh medium and antigen were able to produce MIF. Gel filtration by Sephadex G-100 chromatography indicated that HSF had an approximate m.w. of 23,000 to 40,000. These results suggest that the release of histamine at the sites of immediate hypersensitivity reactions, possibly by generating HSF activity, may play a regulatory role in the subsequent development of cellular-immune reactions at the same site.     

Complement-mediated release of histamine from human basophils. II. Biochemical characterization of the reaction.

Release of histamine from human basophils was induced by activation of complement using zymosan. The histamine-releasing factor resembled C5a on the basis of m.w. (15,000) as well as previous studies showing inactivation by anti-C5. Complement-induced release of histamine was compared with allergic release of histamine which is mediated through appropriate allergens and reaginic IgE. Previously we demonstrated that the former reaction occurred more quickly. Both reactions were inhibited by drugs which increase intracellular concentrations of cAMP3 (theophylline, prostaglandin E1, and histamine) or which mimic the action of cAMP (its dibutyrly derivative). Calcium was required for complement-mediated release of histamine and an increasing response was observed up to physiologic concentrations (2 mM). Magnesium (0 to 1 mM) did not affect the amount of histamine released. Also, glycolysis was probably required for optimal release by complement, since both 2-deoxyglucose and iodoacetamide were inhibitory. When basophils were partly enriched by depletion of neutrophils and eosinophils, the percentage of histamine released by complement was unchanged. Finally, it was shown that activated complement desensitized basophils from responding to a second challenge by the same stimulus. Cross-desensitization was not observed between complement and pollen allergens.     

Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2−/− mice

Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis.MethodsFVB/NJ and Mdr2^−/− mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber.ResultsBiliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2^−/− mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2^−/− mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation.ConclusionCholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.