Demonstration of Histamine Receptors on Human Platelets by Flow Cytometry

Fluoresceinated human albumin conjugated with histamine (FHA-HIS) has been used for the demonstration of histamine receptors on human platelets. Such receptors were demonstrated on 40–63% of peripheral blood platelets in 4 healthy donors. The binding of FHA-HIS was inhibited on 35–79% of the platelets by the histamine H₁ receptor antagonists diphenhydramine and clemastine. The histamine H₂ receptor antagonist cimetidine blocked the FHA-HIS binding on 14–37% of the platelets. It is concluded that histamine H₁ as well as H₂ receptors occur on human platelets but the receptors are not equally distributed in the platelet population.     

Histamine in chronic allergic responses.

In addition to its well-characterized effects in the acute inflammatory and allergic responses, histamine has been shown to affect chronic inflammation and regulate several essential events in the immune response. Histamine can selectively recruit the major effector cells into tissue sites and affect their maturation, activation, polarization, and effector functions leading to chronic inflammation. On the other hand histamine acting through its receptor (HR) type 2 positively interferes with the peripheral antigen tolerance induced by T regulatory (Treg) cells in several pathways. Histamine also regulates antigen-specific TH1 and TH2 cells, as well as related antibody isotype responses. These findings provide suitable explanation for the observations in the experimental model of asthma showing that allergic inflammatory responses and bronchial hyperresponsiveness may be susceptible to HR1 blockade. Apparently, the various effects of histamine on immune regulation are due to differential expression and regulation of 4 histamine receptors and their distinct intracellular signals. In addition, differences in affinities of these receptors is highly decisive on the biological effects of histamine and drugs that target histamine receptors. This article highlights novel discoveries in histamine immunobiology and discusses their relevance to the allergic inflammatory responses.     

Histamine stimulates the proliferation of human articular chondrocytes in vitro and is expressed by chondrocytes in osteoarthritic cartilage

OBJECTIVES: To determine the effects of histamine on the proliferative rate of human articular chondrocytes (HAC) in vitro, and to demonstrate whether HAC in osteoarthritic (OA) cartilage express histamine and histidine decarboxylase (HDC). METHODS: HAC in vitro were incubated with and without histamine in 96 well culture plates and the extent of cell proliferation was determined using the naphthol blue-black method. Histamine effects were analysed with the histamine H(1) and H(2) receptor antagonists, mepyramine and ranitidine, respectively. Rabbit polyclonal antibodies and alkaline phosphatase conjugated secondary antibodies were used, and histamine and HDC were demonstrated by immunohistochemistry in OA cartilage tissues. RESULTS: Histamine stimulated the proliferation of HAC in culture. This stimulation was blocked by the addition of mepyramine, but not ranitidine, suggesting that the effect is mediated through H(1) histamine receptors. The addition of alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase (the enzyme responsible for histamine production), reduced the rate of proliferation of HAC. Both histamine and histidine decarboxylase were demonstrated in chondrocytes of OA cartilage by immunohistochemistry. CONCLUSION: Changes induced by histamine in the proliferative rate of HAC may contribute to the formation of chondrocyte clusters associated with OA cartilage; an observation supported by the demonstration of histamine and HDC expression by chondrocytes of OA cartilage in situ.     

Histamine reverses IL-5-afforded human eosinophil survival by inducing apoptosis: pharmacological evidence for a novel mechanism of action of histamine

Eosinophils are essential inflammatory cells in the pathogenesis of asthma and atopic conditions. Histamine, released from mast cells and basophils in response to allergen exposure, is a critical mediator in the allergic response. Histamine exerts its effects via four unequivocally characterized histamine receptors, H(1-4). Several functions of eosinophils have previously been shown to be stimulated by histamine. However, its effects on eosinophil apoptosis are unknown. The aim of the present study was to resolve the effects of histamine on constitutive apoptosis of human eosinophils and on the survival-enhancing action of interleukin (IL)-5. Additional experiments were conducted to elucidate the histamine receptor(s) involved in any response seen and the associated signal transduction cascade. Human isolated peripheral blood eosinophils were cultured in the absence or presence of histamine, IL-5 and receptor antagonists/agonists or mediator inhibitors/analogues. Apoptosis was assessed by measuring the relative DNA content of propidium iodide (PI)-stained cells and the effects were confirmed by morphological analysis with bright field microscopy. Caspase activities were assessed by using commercial Caspase-Glo 3/7, 8 and 9 luminescence assays. Histamine (10-100 microM) partially reversed IL-5-induced human eosinophil survival by enhancing apoptosis as assessed by measuring the relative DNA content of PI-stained cells. This effect was not mediated through any of the known histamine receptors or through non-specific activation of 5-hydroxytryptamine receptors or alpha-adrenoceptors. Moreover, the reversal of IL-5-inhibited eosinophil apoptosis by histamine seemed not to utilize the conventional intracellular second-messenger pathways including cyclic AMP, protein kinase A or phospholipase C. Inhibition of caspase 6 and caspases 1, 10 or 12 reversed the effects of histamine but also inhibited apoptosis in general. In conclusion, the data presented herein indicate that histamine induces human eosinophil apoptosis in the presence of a survival-prolonging cytokine by a mechanism that does not apparently involve the activation of any of the currently known histamine receptor subtypes. The possibility exists that another, as yet unidentified, histamine receptor may exist in human eosinophils that regulates survival, although the participation of histamine receptor-independent mechanisms cannot be excluded.     

Polymorphonuclear leukocyte histamine receptors: occurrence in cell surface clusters and their redistribution during locomotion.

A univalent and bioactive fluorescent derivative of histamine bound to the surface of human polymorphonuclear leukocytes; free histamine was found to compete with this derivative for binding sites. Histamine H2-receptor specificity was indicated by binding inhibition experiments using cimetidine (H2-specific) but not diphenhydramine (H1-specific). Video-intensification fluorescence microscopy was used to determine the distribution of histamine receptors in living polymorphonuclear leukocytes. Receptors appeared as randomly distributed clusters upon stationary cells. During random locomotion, receptors were restricted to the ends of pseudopods, whereas chemotaxis led to receptor localization at lamellipodia and uropods. Ligand-receptor complexes were restricted to the cell surface, as shown by quenching exterior fluorescence with crystal violet. Therefore, pinocytic uptake cannot account for the observed receptor localization or clustering. As a further control, the lipid analog 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine remained uniformly distributed during all conditions. Histamine-mediated inhibition of adherence may be related to formation of ligand-receptor membrane domains at adherence sites.     

L'histamine,une nouvelle cytokine du lymphocyte T ?

Histamine is a chemical mediator with, in addition to its well-known effects in the acute inflammatory and allergic responses, a less known role in the regulation of the immune response. Indeed, histamine regulates the activation of T cells and notably affects Th1/Th2 balance by interfering with the cytokine network and by controlling their production. However, the numerous studies dealing with the effect of histamine during this control have given rise to several conflicting datas. These discrepancies might primarily be explained by the existence of four types of histamine receptors and their differential function and expression on cells that may therefore vary with the experimental setup. In this review, we thus highlight the complexity of this system and the existence of interactions between histamine, T cells and cytokines.     

Histamine H1 receptors on adherent rheumatoid synovial cells in culture: demonstration by radioligand binding and inhibition of histamine-stimulated prostaglandin E production by histamine H1 antagonists.

Histamine H1 receptors have been demonstrated on adherent rheumatoid synovial cells using biochemical and radioligand binding assays in vitro. The addition of histamine (17.8 mumol/l) to nine primary cultures of adherent rheumatoid synovial cells resulted in a two- to 21-fold increase in the production of prostaglandin E (PGE). This increase was inhibited by three H1 receptor antagonists (mepyramine, tripelennamine, and chlorpheniramine) in a dose related manner at concentrations below 10(-6) mol/l. Competitive binding assays with [3H]mepyramine gave ED50 values of approximately 10(-5) mol/l for the three H1 antagonists. H2 receptor antagonists (cimetidine and ranitidine) did not inhibit the histamine induced increase in PGE and did not compete effectively with the binding of H1 antagonists.     

The Effect of H2 Antagonists on Proliferation and Apoptosis in Human Colorectal Cancer Cell Lines

Cimetidine is known to enhance the survival of gastro-intestinal cancer patients, though the mechanisms involved are incompletely understood. Postulated modes of action include blocking the proliferative effect of tumors and inhibiting T suppressor cell activity, both of which are thought to be mediated by histamine type 2 receptors. Apoptotic cell death may offer an alternative explanation for reduced cell growth. We aimed to examine the effects of histamine, cimetidine, and ranitidine on in vitro proliferation and apoptosis in two human colorectal cancer cell lines, Caco-2 and LoVo. A cell proliferation assay was used as an index of cell growth. Histamine receptor status was determined by quantifying cyclic adenosine monophosphate and apoptosis via DNA fragmentation. Results show that histamine (10(-5) to 10(-9) M) had no effect on the growth of either cell line. The proliferation of Caco-2 was inhibited by ranitidine (10(-7) M) alone and in combination with histamine. Cimetidine (10(-5) M) only suppressed the growth of Caco-2 in the presence of histamine. The H2 antagonists had no effect on LoVo irrespective of histamine. There was no accumulation of cyclic adenosine monophosphate in Caco-2 cells in response to histamine at a similar concentration. Apoptosis was induced in Caco-2 by both antisecretory drugs, and only ranitidine caused apoptotic cell death in LoVo cells. We conclude that cimetidine and ranitidine inhibit Caco-2 cancer cells in vitro, independently of the H2 receptor. In addition, both drugs induce apoptosis in the same cell line. Growth inhibition and apoptosis are likely to contribute to the tumor regressive properties of cimetidine and ranitidine in vivo.     

Molecular cloning of a gene encoding the histamine H2 receptor.

The H2 subclass of histamine receptors mediates gastric acid secretion, and antagonists for this receptor have proven to be effective therapy for acid peptic disorders of the gastrointestinal tract. The physiological action of histamine has been shown to be mediated via a guanine nucleotide-binding protein linked to adenylate cyclase activation and cellular cAMP generation. We capitalized on the technique of polymerase chain reaction, using degenerate oligonucleotide primers based on the known homology between cellular receptors linked to guanine nucleotide-binding proteins to obtain a partial-length clone from canine gastric parietal cell cDNA. This clone was used to obtain a full-length receptor gene from a canine genomic library. Histamine increased in a dose-dependent manner cellular cAMP content in L cells permanently transfected with this gene, and preincubation of the cells with the H2-selective antagonist cimetidine shifted the dose-response curve to the right. Cimetidine inhibited the binding of the radiolabeled H2 receptor-selective ligand [methyl-3H]tiotidine to the transfected cells in a dose-dependent fashion, but the H1-selective antagonist diphenhydramine did not. These data indicate that we have cloned a gene that encodes the H2 subclass of histamine receptors.     

Studies of histamine in the rheumatoid joint

Summary Fragments of synovial membranes from three patients with rheumatoid arthritis were incubated in Dulbecco's modified Eagle's medium (DMEM). Histamine was released into the medium at rates up to 649.3 ng/g synovium/day. To investigate whether histamine in synovial fluid may have more than a vasoactive role, cells cultured from human trabecular bone adjacent to a rheumatoid joint and human adherent rheumatoid synovial cells were incubated with different concentrations of histamine for 5 min. Histamine increased the intracellular content of cyclic AMP up to three fold in a dose-related manner but had no effect on synovial cells. This offers preliminary evidence that cells grown from human trabecular bone have histamine receptors.     

Histamine and immune reactions. 1. Histamine receptor-bearing lymphocytes

Literature review. Mast cells and basophils are included not only in anaphylactic but also in complement activating and cell-mediated immune reactions. A histamine release can be induced via the IgE mechanism, the anaphylatoxins, the histamine releasing activity, and by other reactions. Histamine and several other mediators induce a local inflammation reaction either by a direct action on their target cells or indirectly by the activation of other humoral and/or cellular effector systems. Histamine influences several immune reactions. Here, its influence on histamine receptor-bearing lymphocytes is discussed more in detail. Using in vitro test systems, it has been shown that histamine has a dual effect, it induces a suppression when applied in higher concentrations, while low concentrations are stimulating. In vivo, histamine receptor-bearing lymphocytes have suppressive functions, mediated by H2 receptors. Some preliminary evidence for the inclusion on histamine in the immunoreagulation of patients with several diseases is presented.     

Surrogate thyroglobulin receptors and T cell proliferation in Hashimoto's thyroiditis.

Immunoglobulin molecules on the surface of a B lymphocyte are the endogenous "receptors" to which specific antigens bind. Studies in mice have shown that a monoclonal antibody, conjugated with palmitate to provide a lipid tail, can be inserted into the cell membrane to provide a "surrogate" antigen receptor. We have investigated whether a palmitate conjugate of a human monoclonal antibody specific for thyroglobulin (TG) could function as a surrogate TG receptor on blood mononuclear cells separated into fractions enriched for T cells or depleted of T cells (non-T cells). Using flow cytometry, we detected surrogate TG receptors on non-T (but not on T) cells from 11 of 11 individuals studied (5 Hashimoto patients and 6 control donors). In contrast, endogenous TG receptors could only be detected on non-T cells from 1 of 3 Hashimoto patients and from 0 of 4 control donors. Because of the efficient binding of TG by surrogate receptors on non-T cells, we assessed the ability of such cells to present TG to T cells. Proliferation in response to TG was observed in T cells from only 1 of 5 Hashimoto patients. This low frequency of response was no different from that previously detected using cultures of T cells and autologous dendritic cells. Therefore, the successful generation of surrogate receptors on non-T cells is not associated with more efficient TG presentation of T cells. Furthermore, the significance of the present study is that the T cells, not the antigen-presenting cells, are likely to be the limiting element in the T cell proliferative response to TG and other thyroid autoantigens.     

Histamine containing endocrine cells in the human stomach.

Histamine and chromogranin A-immunoreactive cells were studied in the mucosa of the human stomach. Cells reacting to both histamine and chromogranin A-antibodies (histamine containing endocrine cells), were demonstrated by double immunostaining and found to be restricted to the oxyntic mucosa. Histamine containing cells that did not stain with chromogranin A-antibodies were numerous throughout the stomach. Of the histamine immunoreactive cells in the oxyntic gland area 22% reacted with antibodies against chromogranin A. Histamine containing endocrine cells constituted 44% of the total number of endocrine cells in the oxyntic mucosa. These findings suggest that the histamine containing endocrine cells in the human stomach are identical with the so called enterochromaffin like cells.     

Killing of K562 cells with conjugates between human transferrin and a ribosome-inactivating protein (SO-6)

Cellular iron uptake is mediated by binding of transferrin with specific surface receptors and internalization of the Fe-transferrin-receptor complex. This has been examined as a possible pathway for carrying into leukaemic cells a ribosome-inactivating protein (RIP), SO-6, derived from Saponaria officinalis. Purified human differic transferrin was conjugated with SO-6 and a pool of proteins was obtained, with variable numbers of SO-6 molecules linked to a single transferrin molecule. Human erythroleukaemic K562 cells were grown in the presence of human transferrin, SO-6 and human transferrin conjugated with SO-6. The conjugate was found to be internalized via binding with transferrin receptor. Whereas the presence of unconjugated human transferrin and SO-6 in the medium did not significantly influence K562 cell growth, the conjugated proteins displayed an inhibitory activity on cell proliferation. This was maximal after 72 h at a transferrin concentration of 10(-9) M, with about 50% of cells being killed. Bovine transferrin, present in fetal calf serum, did not appear to compete with human diferric transferrin in binding to K562 cells in suspension culture. In a clonogenic assay, colony formation by leukaemic cells was not influenced by free SO-6 or transferrin, whereas the conjugated proteins were markedly inhibitory (about 100% at 10(-9) M). Our findings indicate that SO-6 can be efficiently carried into mammalian cells via the transferrin-transferrin receptor cycle and exert its ribosome inactivating activity. This is in keeping with the existence of an alternative pathway of transferrin endocytosis in addition to the classic acidic endosome pathway. From a practical viewpoint, conjugates between transferrin and SO-6 can be useful tools for studying the expression of transferrin receptors, and deserve also to be investigated for a possible use in cancer therapy.     

Histamine,récepteurs de l’histamine et anti-histaminiques : données récentes

Histamine was discovered at the beginning of the 20th century. It has been recently shown that immunocompetent cells (T lymphocyte, dendritic cell) are capable of histamine synthesis and that histamine can regulate the Th1/Th2 balance. This immune regulation by histamine is related to its activity as a potent polarizing factor for dendritic cells, giving rise to DC2 dendritic cells that are involved in the differentiation of T helper cells towards Th2 phenotype. The majority of histamine is metabolized by N-methyltransferase. A common polymorphism of this enzyme that is associated with decreased enzyme activity has been associated with asthma. Histamine binds to four receptor types that are expressed on different types of cells and that mediate the numerous actions of histamine. H₁-receptors exhibit constitutive activity that is inhibited by several H₁-receptor antagonists, which therefore display a negative intrinsic activity called inverse agonist activity. In addition, the cerebral P-glycoprotein-mediated efflux of recent H₁-receptor antagonists may explain the lack of nervous system side effects of second-generation anti-histamines.     

Extremely early onset of ranitidine action on human histamine H2 receptors expressed in HEK293 cells

BACKGROUND/AIMS: Histamine H2 receptor antagonists are considered to exert their effects on gastric acid secretion more rapidly than proton pump antagonists. However, there are no reports concerning the direct interaction of a histamine H2 receptor antagonist with the human H2 receptor in terms of onset of action. This study aims to characterize how rapidly famotidine and ranitidine, the most widely used histamine H2 receptor antagonists, interact with the human histamine H2 receptor. METHODS: HEK293 cell lines, stably expressing human histamine H2 receptors, were obtained. The dose- and time-dependent effects of famotidine and ranitidine on [3H]-tiotidine binding and histamine-stimulated cAMP production were analyzed. RESULTS: Ranitidine inhibited both [3H]-tiotidine binding and histamine-stimulated cAMP production more promptly than did famotidine. Inhibition of histamine-stimulated cAMP production by Cmax doses of famotidine (20 mg p.o.) and ranitidine (150 mg p.o.) peaked by 15 and 2 min, respectively. [3H]-tiotidine binding was not saturated by 60 min at the famotidine Cmax, while the ranitidine Cmax had produced saturation by 15 min. CONCLUSION: Ranitidine inhibits the human histamine H2 receptor very rapidly.     

Histamine network in atherosclerosis

Histamine is a low-molecular-weight amine, synthesized from l-histidine by histidine decarboxylase. It has been suggested that the histamine is produced in the atherosclerotic lesion although the activity of histamine has not been clarified completely. To avoid the pharmacologic problems, genetically engineered mice are useful. We recently observed the histidine decarboxylase-gene knockout mice ameliorates' atherosclerotic region, compared with that of the wild-type control mice. The source of histamine in atherosclerotic lesion should be clarified in details; however, it could be macrophage, endothelial cells, and mast cells. All four types of histamine receptors (H1-H4) have the possibilities to be involved in the atherosclerotic regions. Because H1 and H2 receptors are discovered previously, the activities through those receptors are investigated relatively well, but as the other two types of receptors have been cloned recently, their involvement in atherosclerotic lesion should be investigated further.     

A role for histamine receptors in rheumatoid arthritis

Although neurotransmitters and various chemical mediators play an important role in the pathogenesis of rheumatoid arthritis (RA), precise underlying mechanisms have yet to be determined. Histamine is a classical mediator of inflammation and three types of receptors are known. We investigated the presence and functions of histamine receptors of lymphocytes, bone marrow cells, synovial fibroblasts, and chondrocytes in experimentally-induced arthritis and human RA. The function of H₂ receptors in peripheral blood lymphocytes and bone marrow cells were down regulated as measured by increments of intracellular cAMP and IL-6 production. Synovial fibroblasts from RA patients did not respond to H₂ agonist to synthesize hyaluronic acid. It is evident that H₂ receptors are down-regulated in lymphocytes, bone marrow cells, and synovial fibroblasts. The reduced function of H₂ receptors in collagen-induced arthritis was normalized by transfer of the receptor-bearing lymphocytes and bone marrow cells. These data suggest that histamine is involved in the pathogenesis of RA.     

Demonstration of functional oxytocin receptors in human breast Hs578T cells and their up-regulation through a protein kinase C-dependent pathway

Oxytocin (OT) receptors (OTRs) have been demonstrated in a number of human breast tumors and tumor cells, but it was not clear whether the receptors were functional. We examined the regulation and function of OTR in a tumor cell line, Hs578T, derived from human breast. These cells expressed moderate levels of OTR when cultured in 10% FBS, as demonstrated by RT-PCR and binding analyses. Serum deprivation resulted in the loss of OTRs, with no effect on cell viability. Restoration of serum and addition of 1 microM dexamethasone (DEX) increased OTR levels by about 9-fold. Up-regulation was blocked by the addition of phospholipase C and PKC inhibitors. Serum/DEX treatment also increased steady state OTR messenger RNA levels. OT increased intracellular Ca2+ in a time- and dose-responsive manner, and the effects of OT were lost when OTRs were down-regulated by serum starvation. Serum/DEX up-regulation of OTR restored the responsiveness to OT. OT also stimulated ERK-2 (extracellular signal-regulated protein kinase) phosphorylation and PGE2 synthesis in Hs578T cells. In addition to showing that OTRs in the breast tumor cells are functional, these studies show that Hs578T cells can be used to study molecular regulation of OTR gene expression and intracellular signaling pathways stimulated by OT.     

Effects of N-alpha-methyl-histamine on human H(2) receptors expressed in CHO cells

BACKGROUND: Production of N-alpha-methyl-histamine (NAMH), a histamine H(3) receptor (H3R) agonist, is reportedly promoted in Helicobacter pylori infected human gastric mucosa. NAMH was suggested to act directly on histamine H(2) receptors (H2Rs) in animals to stimulate acid secretion and to be a H2R agonist. As H2Rs and H3Rs play different roles in gastric acid secretion, it is very important to verify that NAMH is a H2R agonist. AIMS: To determine whether NAMH is a H2R agonist, as well as a H3R agonist. METHODS: We used a Chinese hamster ovary (CHO) cell line expressing human H2Rs (CHO-H2R) and control CHO cells. Expression of human H2Rs was confirmed by tiotidine binding. cAMP production in CHO-H2R and control cells in response to histamine or NAMH was measured. cAMP production in response to 10(-7) M NAMH was also measured in the presence or absence of the H2R antagonist famotidine and the H3R antagonist thioperamide. RESULTS: NAMH dose dependently stimulated cAMP productions in CHO-H2R cells. This production was inhibited by famotidine but not by thioperamide. Control CHO cells were unresponsive to either histamine or NAMH. In addition, the effect of NAMH, in terms of cAMP production in CHO-H2R cells, was more potent than that of histamine-that is, with a lower EC(50) concentration and higher maximal cAMP production. Both NAMH and histamine, but not R-alpha-methyl-histamine, effectively inhibited [(3)H] tiotidine binding to CHO-H2R cells. CONCLUSIONS: NAMH, which is produced in the gastric mucosa by H pylori, is a potent H2R agonist as well as a H3R agonist.