Chronic morphine accelerates the progression of lipopolysaccharide-induced sepsis to septic shock

Abstract

Opiate addicts have been shown to have a high susceptibility to bacterial infection. We investigated how treatment with morphine alters lipopolysaccharide (LPS)-induced inflammatory responses in the rat. Chronic morphine alone elevated serum endotoxin levels. Animals treated with morphine and LPS (250 μg/kg) developed hypothermia, decreased mean arterial pressure (MAP), increased plasma thrombin anti-thrombin III (TAT) complex, and approximately 67% of animals exhibited progressive intramicrovascular coagulation. Morphine also enhanced LPS-induced leukocyte–endothelial adhesion (LEA), suppressed leukocyte flux, and corticosterone production, and elevated interleukin-1β, tumor necrosis factor-α, and interleukin-6 serum levels. Our study presents both the molecular and cellular mechanisms underlying the potentiated LPS-induced inflammation and accelerated progression to septic shock seen with chronic morphine exposure.

Introduction

Morphine is an opiate commonly used for analgesic purposes. Morphine's addictive properties make it subject to abuse. Morphine tolerance begins with the initial treatment and becomes apparent after 2–3 weeks with regular therapeutic doses (Katzung, 1992). It has been known for decades that this opiate-tolerant state associates with immunological consequences. Studies show that morphine suppresses lymphocyte trafficking (Flores et al., 1995), the lymphocyte proliferative response to mitogens Bryant et al., 1991, Fecho et al., 1995, natural killer T-cell activity Gomez-Flores and Weber, 1999, Yokota et al., 2000, the production of antibodies (Bussierre et al., 1993), the total number of circulating leukocytes LeVier et al., 1994, Fecho and Lysle, 2002, atrophy of the spleen and thymus, and enhances endotoxemia Patel et al., 1996, Roy et al., 1998, Roy et al., 1999.

Morphine-induced immunosuppression subsequently increases patient susceptibility to bacterial infection (Hilberger et al., 1997). Lipopolysaccharide (LPS) is a component of Gram-negative bacteria that is shed from the outer cell wall and is a potent endotoxin (Lukasiewicz and Lugowski, 2003). While LPS has no direct toxicity, it leads to the activation of the complement system and the coagulation cascade, and induces immune cells, such as macrophages and neutrophils, to release proteins termed endogenous mediators of sepsis (Parrillo, 1990). The term, sepsis, refers to a bacterial infection that produces a systemic immunological response. Septic patients suffer from either high or low body temperature, and an elevated heart rate, respiratory rate, and white blood cell count. At the molecular level, tumor necrosis factor-alpha (TNF-α), which is released from macrophages, is the primary mediator of sepsis by increasing vascular permeability, enhancing leukocyte–endothelial interaction, and activating platelets (Parrillo, 1993). In addition, TNF-α stimulates macrophages and endothelial cells to release interleukin-1 (IL-1), an endogenous pyrogen which acts on the hypothalamus causing the fever usually associated with bacterial infections. IL-1, in turn, triggers the release of other pro-inflammatory cytokines, such as interleukin-6 (IL-6), and prostaglandins Ato et al., 2002, Li et al., 2002, Itoh et al., 2003. Initially this cascade of events is protective, acting to contain and eliminate the infection. However, as the bacteria continue to proliferate and exude increasing amounts of endotoxin, sepsis can progress to septic shock Wang et al., 2003, Asakura et al., 2003.

Septic shock is a deadly response to bacterial infection. The mortality rate is high, and it is one of the most common causes of death in the intensive care unit (Wheeler and Bernard, 1999). Septic shock causes a dramatic decrease in blood pressure and the onset of disseminated intravascular coagulation (DIC). The hypotension is caused by an excessive increase in vascular permeability, vasodilation, and decreased peripheral resistance (Wheeler and Bernard, 1999). DIC ensues due to activation of the coagulation cascade via the Hageman factor (factor XII), leading to thrombosis, tissue ischemia, and eventual multi-organ failure Parrillo, 1993, Yamaguchi et al., 2000. Another complication of DIC is excessive bleeding and hemorrhage through the depletion of platelets and anti-coagulant factors, such as anti-thrombin III. In the pathogenesis of septic shock, while the concentration of endotoxin or LPS is important, it is the inflammatory response to the endotoxin that is the key factor in determining the progression of the disease.

Inflammation is a critical component of innate immunity, being one of the initial responses to invading pathogens. Inducing functional leukocyte–endothelial interaction (LEI) is essential for the generation of an effective inflammatory response (Eppihimer and Granger, 1997). Under normal conditions, LEI is minimal, with the majority of erythrocytes and leukocytes being restricted to the lumen of the blood vessel House and Lipowsky, 1987, Nazziola and House, 1992, Rosenkranz and Mayada, 1999. The incidence of LEI increases, as a result of vascular injury or an infectious challenge, such as with endotoxin, vasoactive peptides, chemoattractant factors, and pro-inflammatory cytokines. The process of LEI includes leukocyte rolling on the vascular endothelium, leukocyte adhesion, and leukocyte extravasation out of the lumen along a chemotactic gradient towards the infecting pathogen Muller et al., 1993, Salas et al., 2002, Smith et al., 2002. A functional LEI response to infection would consist of an increase in leukocyte–endothelial adhesion (LEA) accompanied by a decrease in leukocyte flux (FLUX). This model has been used as an indicator of an intact and functional immune response. However, few studies have successfully examined the LEI response during the development of DIC due to the exacerbated LEA, decreased leukocyte flow, and systemic coagulation. Studies of progressing DIC become even more complex when the immunomodulatory systems are compromised.

While the inflammatory response is necessary for the containment and clearance of infections and the healing of injuries, regulatory mechanisms must be in place to ensure that the inflammatory response is not excessive. A major pathway through which the central nervous system regulates the immune system is the hypothalamic–pituitary–adrenal (HPA) axis (Bateman et al., 1989). The HPA axis controls the hormonal stress response, the mechanism by which the CNS exerts its modulatory actions on the immune response (Sternberg et al., 1992). The pathway originates within the paraventricular nucleus (PVN) of the hypothalamus. The PVN secretes corticotropin-releasing hormone (CRH) into the hypophyseal blood supply, which stimulates the anterior pituitary to release adrenocorticotropic hormone (ACTH) Katsuura et al., 1988, McCoy et al., 1994, Lacosta et al., 1998. The released ACTH induces the adrenal glands to synthesize and release glucocorticoids, i.e., cortisol in the human and corticosterone in the rat (Hansen et al., 2000). Glucocorticoids, such as corticosterone, exert powerful anti-inflammatory effects. Previous studies have shown that chronic morphine treatment desensitizes the HPA axis, leading to desensitization of IL-1β-induced activation of FOS immunoreactivity in the PVN (Chang et al., 1996) and potentiation of IL-1β-induced LEA in the rat mesentery (House et al., 2001). Chronic morphine treatment has also been shown to induce sepsis in an animal model Hilberger et al., 1997, Roy et al., 1998. Furthermore, morphine synergizes with LPS in the progression of endoxemia (Roy et al., 1999).

In this study, we demonstrated that rats chronically treated with morphine and given a subsequent intraperitoneal (i.p.) injection of LPS showed increased LEI, suppressed production of corticosterone, and elevated levels of TNF-α, IL-1β, and IL-6. In addition, LPS-challenged rats treated with chronic morphine developed a decrease in MAP, reduced body temperature, and plasma TAT concentrations significantly greater than placebo-treated rats. Chronic treatment with morphine alone resulted in an increase in serum endotoxin levels. Taken together, these data provide insight into the physiologic phenomena associated with the progression of sepsis in morphine-tolerant individuals at both the molecular and cellular levels.