Substance P augments interleukin-10 and tumor necrosis factor-α release by human cord blood monocytes and macrophages

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Abstract

We have investigated the effects of SP on the constitutive and/or lipopolysaccharide (LPS)-induced expression of interleukin-10 (IL-10) and tumor necrosis factor (TNF-α) in both freshly isolated cord blood monocytes (FICBM) and cord blood monocyte-derived macrophages (CBMDM). The cells were treated with SP at various concentrations (10−14 to 10−6 M) in the presence or absence of LPS and culture supernatants were analyzed for IL-10 and TNF-α as measured by an enzyme immunosorbent assay (ELISA). FICBM and CBMDM treated with SP alone increased TNF-α secretion. The stimulatory effects of SP on TNF-α secretion are inhibited by a anti-SP polyclonal antibody and SP antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-9-Leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the SP receptor). Although the treatment with SP alone did not enhance IL-10 secretion by both freshly isolated and cultured cord monocytes, treatment with SP in combination with LPS leads to a synergistic interaction in upregulation of IL-10 secretion. Fragments of SP (SP1–4 and SP5–11) in the presence or absence of LPS show little effects on IL-10 secretion by FICBM. SP reverses the inhibitory effect of IFN-γ on LPS-induced IL-10 secretion by FICBM. In addition, the two SP antagonists and the anti-SP polyclonal antibody blocked the SP effect on IL-10 secretion by FICBM, indicating that these effects are specific and SP receptor mediated. Thus, SP is likely to play an important role in certain inflammatory conditions in the immune and nervous systems.

Introduction

Monocytes and macrophages regulate many aspects of immune function including antigen processing, T cell proliferation, antibody production, tumor defense and cytokine production (Auger and Ross, 1992; Ho and Douglas, 1995). Monocytes secrete proinflammatory cytokines which include IL-1, IL-6, TNF-α and IL-12 (Auger and Ross, 1992; Ho and Douglas, 1995). They also produce interleukin-10 (IL-10) which inhibits synthesis of proinflammatory cytokines and are essential elements of the anti-viral responses, including interferon-gamma (IFN-γ) and IL-2 synthesis by Th1 cells (de Waal Malefyt et al., 1991a; Haward and Garra, 1992). These monokines act in an autocrine and paracrine manner. IL-10 is a pleiotropic molecule possessing both inhibitory and stimulatory activities on cells of the immune system. Elevated levels of IL-10 have been reported in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) of HIV-1 infected individuals as compared to healthy control (Clerici et al., 1994). IL-10 inhibits HIV-1 replication in cells of monocyte/macrophage lineage (Saville et al., 1994; Masood et al., 1994). The inhibitory effect of IL-10 was correlated with a block in endogenous TNF-α and IL-6 secretion from HIV-1 infected monocyte-derived macrophages (Weissman et al., 1994). There is spontaneous production of IL-10 by peripheral blood lymphocytes in HIV-1 infected individuals which may be significant in reducing viral load in vivo (Diaz-Mitoma et al., 1995). IL-10 also plays an important role in cross-regulation of Th1 and Th2 responses (Mosmann and Moore, 1991). IL-10 suppresses the activation, proliferation, and cytokine secretion from CD4+ T cells and Th0, Th1 and Th2 clones (Del Prete et al., 1993; de Waal Malefyt et al., 1993; Taga and Tosato, 1992; Taga et al., 1993). IL-10 acts on monocytes and macrophages by inhibiting TNF-α, IL-1α and β, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion (de Waal Malefyt et al., 1991b).

Substance P (SP), the most extensively studied member of tachykinin family, is a neurotransmitter in the conduction of nociceptive stimuli, and a modulator of neuroimmunoregulation (Payan, 1989; Black, 1994; Gilbert and Payan, 1991). In the nervous system SP is a neurotransmitter and as a mediator of neurogenic inflammation. SP may also play a role in the immune responses and in chronic inflammation. The treatment of monocytes/macrophages with SP leads to several functional events which include generation of thromboxane A2, superoxide, down regulation of membrane-associated 5′ nucleotidase, and stimulation of the synthesis and release of arachidonic acid and its metabolites (Hartung and Toyka, 1983; Hartung et al., 1986). Lucey et al. (1995)have demonstrated SP receptors on in vitro cultured human peripheral blood monocytes and monocyte-derived macrophages. SP stimulates human peripheral blood monocytes to produce the inflammatory cytokines including interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) which are important constituents of immune cell activation that act as physiological inductive signals in the regulation of immune responses (Laurenzi et al., 1990; Lotz et al., 1988). TNF-α is an major mediator of inflammation which upregulates HIV-1 expression in T cells and monocytes in vitro. We have recently demonstrated that SP enhances TNF production from macrophages derived from adult human peripheral blood monocytes (Lee et al., 1994) and that SP modulates HIV-1 replication in human peripheral blood monocyte-derived macrophages (Ho et al., 1996).

The effects of SP on TNF-α and IL-10 secretion by placental cord blood-derived monocytes and macrophages have not been investigated. Since SP influences inflammatory processes by stimulating local macrophages to produce proinflammatory cytokines such as IL-1, IL-6, and TNF-α and IL-10 inhibits the macrophage's ability to produce these cytokines, we studied the effects of SP on production of TNF-α and IL-10 by both freshly isolated cord blood monocytes (FICBM) and cord blood monocytes-derived macrophages (CBMDM). We have demonstrated that SP alone or in a synergistic fashion with LPS enhances TNF-α and IL-10 secretion in these cells, supporting the concept that neuropeptides such as SP are important regulators of cytokine expression in human placental cord blood-derived monocytes and macrophages. These studies provide further evidence for the existence of neuroimmune axis which is regulated by a network of interacting cytokines and neuropeptides.

Section snippets

Isolation and culture of cord monocytes/macrophages

Monocytes were purified from cord blood according to our previously described technique (Hassan et al. (1986), Hassan et al. (1990)). Placental cord blood was obtained from placentas of healthy (37–40 weeks) pregnant mothers. In brief, heparinized cord blood (20–50 U/ml) was obtained from the umbilical vein of healthy term placental following uncomplicated pregnancies and deliveries. The cord blood was diluted with Hank's buffer (1:2) and then separated by centrifugation for 45 min over

TNF-α secretion in SP treated cord monocytes and macrophages

Cord monocytes were prepared as described in Section 2and treated with different concentrations of SP (10−14 to 10−6 M). Very low (20–60 pg/ml) TNF-α secretion was detected in cultured CBMDM by ELISA assay (Fig. 1A). However, when the CBMDM were treated with SP at different concentration (10−14 to 10−6 M) increased TNF-α secretion was observed (Fig. 1A). Although freshly isolated cord monocytes produce endogenous TNF-α (250–500 pg/ml), an increased secretion of TNF-α by the cells treated with

Discussion

The mononuclear phagocytes produce a wide array of potent mediators and cytokines which modulate the inflammatory response (Auger and Ross, 1992; Ho and Douglas, 1995; Nathan, 1987). The nervous system modulates immunologic and inflammatory responses, most likely through neuropeptides such as SP. Lotz et al. (1988)have shown that SP and SK induce the release of IL-1, TNF-α, and IL-6 in freshly isolated adult human monocytes (24–48 h in vitro). We have demonstrated that SP stimulates TNF

Acknowledgements

The authors are grateful to Joann Cutilli for technical assistance. This investigation was supported by NIH-MH No. 49981. The Clinical Research Center of the Hospital of University of Pennsylvania (NIH MO1 RR 00040) made possible collection of placental cord blood samples.

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