Endogenous substance P mediates cold water stress-induced increase in interleukin-6 secretion from peritoneal macrophages.

Previous studies from this laboratory had shown that exposure of mice to cold water stress leads to an increase in the secretion of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha) from their peritoneal macrophages. We now report that the secretion of IL-6 from peritoneal macrophages is also increased after cold water stress and that the peptide substance P (SP) participates in this stress-induced response. The stress paradigm involved subjecting male C57BL/6J mice to 5 min swim tests in 10 +/- 2 degrees C water twice daily for 4 d. Cold water stress augments the lipopolysaccharide-induced IL-6 secretion from peritoneal macrophages, elevates immunoreactive SP (iSP) in the peritoneal wash fluid, and reduces iSP in certain peritoneum-containing tissues or organs (i.e., diaphragm, abdominal wall, ileum, and rectum). The 10 d stress time studies indicate that increased IL-6 secretion is positively related to elevated iSP in the peritoneal wash fluid and inversely related to reduced iSP in certain peritoneum-containing tissues. Pretreatment with capsaicin, which depletes SP in the sensory nerve endings, eliminates stress-control differences in the peritoneal wash fluid and in certain peritoneal tissues. Moreover, RP67,580, a specific SP antagonist, eliminates the cold water stress-induced augmentation of IL-6 secretion from peritoneal macrophages. These results suggest that cold water stress promotes the release of SP from peritoneal tissues into the peritoneal cavity, where it participates in the cold water stress-induced macrophage functional alterations.

Substance P primes murine peritoneal macrophages for an augmented proinflammatory cytokine response to lipopolysaccharide.

We have recently shown that substance P (SP) participates in the stress-induced modulation of elicited, peritoneal macrophage function. This study reports the in vitro effects of SP on macrophage activity. We show by an MTT bioassay that SP significantly increases cellular metabolic activity. We show by ELISA that preincubating (priming) the macrophages with SP, prior to the incubation with lipopolysaccharide (LPS), results in a significant enhancement of proinflammatory cytokine secretion, relative to LPS alone. Finally, we show that somatostatin can antagonize the SP-induced enhancement of cytokine secretion. The above results demonstrate the importance of the temporal sequence in which stimuli are administered, in vitro, and indicate that SP can act as first signal in the cascade of macrophage activation. We postulate that stress, via the secretion of SP and other sensory neuropeptides, may play a role in the pathogenesis of certain inflammatory diseases of unknown etiology.

Substance P and stress-induced changes in macrophages.

The present paper further links nervous-endocrine-immune systems by describing influences of SP on the immune system, and more specifically, on macrophage function. We have discussed how macrophages are important to immune responses in that much of cellular and humoral responses depend on macrophage function. Macrophages are sensitive to stress in that cold-water stress causes increased cytokine production, either spontaneously (IL-1), or after induction with LPS (IL-6, TNF alpha). Increased cytokine levels (IL-1, IL-6) may induce acute phase reactants in the liver, which is presumably the mechanism operative in the studies indicating increases in acute phase reactants after certain stressors in animals. SP is a likely candidate to affect immune function. Previous data show that macrophages from various species have receptors for and respond to SP in vitro. SP stimulates phagocytic and chemotactic capacity, as well as increased cytokine, PGE2, and thromboxane B2 production. SP is also involved in neurogenic inflammation and is likely to be involved in the pathogenesis of several inflammatory diseases. Present data indicate SP's involvement in macrophage responses to stress. We have shown that stress induced differential SP receptor binding to peritoneal macrophages, although the precise nature of binding differences has not yet been clearly elucidated. Stress also induces more immunoreactive SP in the peritoneal fluid that bathes the peritoneal macrophages. We hypothesize that the two events, altered SP binding and concomitant increased ligand, are causally related. In addition to other correlational data showing concomitant increased SP binding plus ligand concentrations, there is more direct evidence that SP ligand may induce SP receptor expression since the SP antagonist, CP-96,345, prevents the induction of SP receptor mRNA in the staphylococcal toxin A-induced gastroenteritis (C. Pothoulakis and S. E. Leeman, personal communication). Further supporting our notion for a causal relationship we have found the elimination of SP in vivo (via capsaicin pretreatment) reduced SP binding, as has been previously reported. We have also examined the role of SP on stress-induced altered macrophage function in vitro. SP greatly enhanced the LPS-induced macrophage TNF alpha production from stressed animals; in contrast, it produced relatively little effect on macrophages from control animals. Capsaicin pretreatment diminished the enhanced cytokine production in response to stress, such that levels of TNF alpha and IL-6 approximated those of control mice. Taken together, past and present data suggest that (1) stress may initiate, or at least contribute to, an inflammatory response, and that (2) SP is involved in the macrophage stress response. SP has long been known to be involved in inflammatory processes; our data further suggest its role in mediating stress-induced cytokine alterations.