Manzoor M. Khan

Beta-adrenergic receptors on human suppressor, helper, and cytolytic lymphocytes

Using the radioligand beta-adrenergic blocker [125I]cyanopindolol (ICYP), we have characterized the beta-adrenergic receptors on Leu 3+ (T helper [TH]), Leu 2+, 9.3- (T suppressor [Ts]) and Leu 2+, 9.3+ (T cytolytic [Tc]) subsets of human lymphocytes. Peripheral blood T cells were isolated by rosetting, and then subsets were purified by their affinities to monoclonal antibodies against their Leu 3 and 9.3 markers. ICYP binding to the subsets was saturable with time and with concentration; the binding was stereoselective and reversible by beta-adrenergic antagonists. A biological response produced by beta agonists increased intracellular concentrations of cAMP and corresponded to the number of binding sites. Each subset of cells had a number of binding sites, which was characteristic for the given subset. The data indicate that the density of distribution of beta-adrenergic receptors was not homogeneous on the precursors of phenotypically and functionally distinct T cells (Ts approximately 2900, Tc approximately 1800 and TH approximately 750 binding sites). The displacement studies using beta-adrenergic agonists were performed on the cytolytic and suppressor T cell subsets, suggesting that the receptors were mainly of the beta-2 type. The immunobiological significance of such selective distribution of numbers and subtypes of beta-adrenergic receptors on distinct T cell subsets is under investigation.

Effects of histamine on Th1/Th2 cytokine balance

Atopic asthma is a chronic inflammatory disorder of the airways where upon exposure to allergens, the body mounts an immune response. This disease is associated with an increase in the number of Th2 (T helper type 2) cells and Th2 cytokines and a decrease in the number of Th1 (T helper type 1) cells and Th1 cytokines. Histamine plays an important role in the pathogenesis of atopic asthma through differential regulation of T helper lymphocytes. Histamine enhances the secretion of Th2 cytokines such as IL-4 (interleukin-4), IL-5, IL-10 and IL-13 and inhibits the production of Th1 cytokines IL-2 and IFNgamma (interferon-gamma) and monokine IL-12. It has been shown that histamine can modulate the cytokine network through upregulation of PGE(2) (prostaglandin E(2)) and NO (nitric oxide). Histamine also affects cytokine production via H2 receptors and through the activation of PKA (protein kinase A). We have also demonstrated that the Jak-STAT (Janus kinase-signal transducers and activators of transcription) pathway is involved in histamine-mediated regulation of Th2 cytokines IL-5, IL-10, IL-13 and Th1 cytokine IFNgamma. While standard treatment of asthma consists of beta-receptor agonists and inhaled corticosteroids, the elucidation of histamines control over the cytokine network and the Th1/Th2 balance provides a basis for the potential use of antihistamines in the prevention and treatment of atopic asthma. Several other anti-allergic agents to modulate the Th1/Th2 balance are under current investigation based on this paradigm. These include cytokines, cytokine antagonists, anti-IgE, and vaccinations. As more advances are made in our understanding of histamine and its control over the Th1/Th2 balance, the use of new therapeutic targets such as these will play a prominent role in disease management.

Regulation of interleukin-10 secretion by histamine in TH2 cells and splenocytes

Interleukin-10 is a potent suppressive factor that down-regulates cellular immune response via inhibition of the production of TH1 cytokines. Histamine shifts the TH1/TH2 balance from TH1 to TH2 cytokines making the effects of histamine on IL-10 secretion an important factor in this switch. This study was designed to assess the role of histamine in the regulation of IL-10 production and the involvement of PKA and STAT factors in this process. TH2 cells (D10.G4.1) and AKR/j splenocytes were pretreated with histamine at a concentration range of 10(-8)-10(-5) M for 1 h and then activated with PMA + ionomycin or anti-CD3 for 24 h. The supernatants were collected and tested for IL-10 content by ELISA. Histamine stimulated IL-10 production in TH2 cells in a dose-dependent manner that was reversed by both H1- and H2-receptor antagonists and by PKA inhibitors H8 and Rp-cAMPS. Tyrphostin also reversed the stimulation of IL-10 secretion by histamine, indicating that STAT factors were involved in this process. The up-regulation of IL-10 production by histamine in splenocytes was accompanied by inhibitory effects of histamine on IFN gamma production. The pretreatment of splenocytes with histamine in the presence of anti-IL-10 abrogated histamine-mediated inhibition of IFN gamma production suggesting that the effects of histamine on IFN gamma secretion were regulated by IL-10 in multi-cell system.

Phosphodiesterase inhibitors suppress proliferation of peripheral blood mononuclear cells and interleukin-4 and -5 secretion by human T-helper type 2 cells.

It has been suggested that interleukin-4 and -5 (IL-4 and IL-5) are instrumental in the control of allergic disease. Elevated levels of IL-4 messenger RNA (mRNA) have been detected in numerous foci of atopic activity, including bronchoalveolar lavage (BAL) fluid from atopic asthmatics and skin of atopic dermatitis patients. IL-5 is important in eosinophil activation, which is a common feature of atopic disease. IL-5 mRNA has been detected in BAL fluid from both atopic and non-atopic asthmatics, indicating that IL-5 may be a common feature of the two disease states. Production of IL-4 and IL-5 by T cells appears to be associated with a high affinity cyclic AMP (cAMP) phosphodiesterase (PDE). This study was designed to compare the effects of PDE inhibitors Ro20-1724 and theophylline on (1) the mitogenic response of peripheral blood mononuclear cells from atopic and non-atopic individuals and (2) secretion of IL-4 and IL-5 by TH(2) cells after activation with PMA and anti-CD3. Both Ro20-1724 and theophylline inhibited proliferation of PBMC in a dose-dependent manner. There was no significant difference between proliferation of PBMC from atopic versus non-atopic donors, but Ro20-1724, a specific PDE IV inhibitor, was more potent at a concentration of 10(-5)M than theophylline in suppressing lymphocyte proliferation. Similarly, both PDE inhibitors suppressed secretion of IL-4 and IL-5 from TH(2)-like cell lines in a dose-dependent manner. In conclusion, as Ro20-1724 and theophylline inhibit proliferation of PBMC and secretion of IL-4 and IL-5 from human TH(2) cell lines, the development of a selective cyclic nucleotide PDE IV inhibitor may provide a promising new approach for asthma prophylaxis.

Histamine and its lymphocyte-selective derivatives as immune modulators

Abstract Histamine has long been known as a neurotransmitter, inflammatory mediator and a factor in anaphylaxis, cardiac and gastrointestinal functions. Theoretically, it should not be surprising to find that a mediator of inflammation may modulate immunity because receptors for histamine are non-randomly distributed on lymphocytes. Kenneth Melmon and Manzoor Khan cite recent data primarily developed in in-vitro models of delayed hypersensitivity that demonstrate the immune modulatory role of histamine. New lymphocyte-specific histamine H 1 and/or H 2 agonists make it pharmacologically feasible to assess the potential of histamine as a selective in-vivo immune modulator.

Role of Cytokines

This chapter described the physiologic, pharmacologic, and pathologic effects of cytokines. They are glycosylated small polypeptides, synthesized and released by a variety of sources including lymphocytes, leukocytes, and other cell sources. Cytokines are a crucial component of immune response, as they aid in the intercellular communication and are involved in both health and disease, through their physiological and pathological effects. They are involved in mediating natural immunity and regulating the activation, growth, and differentiation of lymphocytes, leukocytes, as well as other cell types. Furthermore, they play a role in the regulation of immune-mediated inflammation and stimulate hematopoiesis. Their effects are mediated via cell surface receptors. Based on their physiological and pathological effects, they are ideal therapeutic agents, either by themselves or as antagonists, which also include specific monoclonal antibodies directed against them.

Histamine regulates the generation of human cytolytic T lymphocytes.

Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.

The effects of histamine on interferon gamma production are dependent on the stimulatory signals.

Histamine regulates the immune response by enhancing TH2 cytokine production and by inhibiting TH1 cytokine production. We assessed the mechanisms of histamines action on helper T cell subsets by evaluating the role of protein kinase A (PKA) in the histamine-mediated effects on IFN gamma production. The splenocytes and TH1 murine cloned cells (pGL10) were pretreated with histamine at a concentration range of 10(-8)-10(-5) M for 1 h and then were activated with anti-CD3, PHA, PMA + ionomycin, or ionomycin for 24 h. The levels of IFN gamma were measured in the supernatants by ELISA. The inhibitory effects of histamine were the most prominent in anti-CD3-stimulated splenocytes (61%). The effects of histamine on IFN gamma production from TH1 cells depended on the mode of cell activation. The activation of cells with anti-CD3 resulted in 27% inhibition of IFN gamma production whereas the activation with ionomycin produced 70% suppression. The inhibitory effects of histamine were completely reversed by cimetidine in a dose-dependent manner in both TH1 cells and in splenocytes. PKA played a role in the inhibition of IFN gamma by histamine when the cells were activated via TCR, and the PKA inhibitors Rp-cAMPS (10(-5) M) and H8 (10(-5) M) reversed the inhibitory effects of histamine on IFN gamma production. However, when the cells were stimulated with ionomycin, the PKA inhibitors did not affect histamine-mediated suppression of IFN gamma production.

Involvement of protein kinase A in histamine-mediated inhibition of IL-2 mRNA expression in mouse splenocytes

The release of histamine from mast cells and basophils during allergic reactions can regulate functions of T cells and may influence the nature of the immune response to a given antigen. The effects of histamine on T lymphocytes are associated with its binding to H2-receptors linked with adenylate cyclase, elevation of cAMP levels and activation of cAMP-dependent protein kinase (PKA). In this report we explore the role of PKA in histamine-mediated effects on IL-2 mRNA expression and IL-2 protein secretion. Fresh isolated mouse splenocytes (C57Bl/6) were pretreated with histamine (10(-4) M) for 1 h in the presence or absence of Rp-cAMPS (50 microM), an inhibitor of PKA regulatory subunit. The cells were then washed thoroughly and activated with plate-bound anti-CD3 (5 microg/ml), or PHA (1:100) or PMA + ionomycin (10 ng/ml, 1 microg/ml) for 6 h. Pretreatment with histamine inhibited IL-2 mRNA expression and secretion in cells activated with anti-CD3 or PMA, but not in cells activated with PMA + ionomycin. Rp-cAMPS prevented histamine-mediated suppression and did not itself affect IL-2 production. These results provide evidence that histamine affected IL-2 production when the cells were activated via the T cell receptor (TCR)/CD3 complex, but did not interfere with signal transduction pathways downstream of PKC leading to production of IL-2. These effects of histamine on IL-2 secretion and mRNA expression were mediated via PKA.

Regulatory effects of mast cells on lymphoid cells: the role of histamine type 1 receptors in the interaction between mast cells, helper T cells and natural suppressor cells

We have investigated the role of mast cells as modulators of lymphocyte function because the mast cells are concentrated in the areas of lymphoid storage; they are dependent upon T-cell growth factor for their proliferation; and they appear to be the principle if not sole storage site for histamine. We have tested the influence of mast cells on the proliferation of alloreactive cloned helper T cells, mixed leukocyte reactions, and the suppressive capacity of natural suppressor cells. We used an IL-3-dependent mast cell line that at high numbers (greater than 10(5)) suppressed and at low numbers (10(3) to 6 X 10(4)) augmented the proliferation of TH cells. Addition of histamine to cocultures enhanced the mast cell mediated proliferation of TH cells without directly affecting the helper cells. The action of histamine appeared to be mediated with H1 type receptors on these mast cells. Pretreatment of natural suppressor cells with supernatants from mast cell enhanced their suppressive capability. Here too, histamines enhanced suppression by the NS cell via histamine type 1 receptors on the natural suppressor cells. Our data suggest that mast cells may be a major modulator of the lymphoid cell immune function and demonstrate a role of histamine type 1 receptors in the interaction between mast cells, helper T cells, and natural suppressor cells.