Conjunctival epitheliopathy induced by topical exposure to bacterial peptidoglycan, muramyl dipeptide.

Noninfectious exudative conjunctivitis can be experimentally produced in rabbits by application of the apoptogenic bacterial cell wall peptidoglycan, muramyl dipeptide (MDP) to the ocular surface. The purpose of this study was to investigate the acute conjunctival cytopathology induced by unilateral ocular surface exposure to MDP. Hematoxylin and eosin staining assessed bilateral tear cytopathology and conjunctival histopathology. The caspases levels in conjunctival tissue and tears were measured in standard assays utilizing p-nitroanaline tagged caspase-specific substrates. Immunofluorescent antibody identified intracellular caspase-3, nuclear factor-κß (NF-κß), and oxidative DNA damage (8-OHdG; 8-oxo-2'-deoxyguanosine) in tear and conjunctiva cells. DNA extracted from conjunctival tissues and pooled tear fluids were visualized by ethydium bromide agarose gel electrophoresis. Onset of ipsilateral conjunctivitis was due to an epitheliopathy characterized by loss of conjunctival epithelial cell adherence, exuviation of conjunctival epithelial cells, and neutrophil infiltration. Caspase-3 levels were significantly higher in exuviated cells in ipsilateral than contralateral tear (p's ≤ 0.001) collected at 3-5 h post MDP. Significantly higher caspase-2, -3, -6, -8 and -9 (p's ≤ 0.03) levels were detected in ipsilateral than contralateral conjunctival tissue at 5 h. Polymeric DNA was detected in ipsilateral but not contralateral conjunctival tissue and tears. Caspase-3, NF-κß, and 8-OHdG positive neutrophils were detected in bilateral conjunctiva and tear. The caspase-3/NF-κß epithelial cells and polymeric DNA in conjunctival tissue and shedding of caspase positive cells and polymeric DNA into ipsilateral tears support MDP induction of acute programmed cell death in vivo. The results suggest that ipsilateral exudative conjunctivitis is due to acute caspase-mediated conjunctival epitheliopathy induced by topical exposure to the bacterial peptidoglycan MDP.

Nod2-Rip2 Signaling Contributes to Intestinal Injury Induced by Muramyl Dipeptide Via Oligopeptide Transporter in Rats.

BACKGROUND AND AIMS: PepT1 can transport bacterial oligopeptide products and induce intestinal inflammation. Our aim was to investigate the mechanism of the small intestine injury induced by bacterial oligopeptide product muramyl dipeptide (MDP) which is transported by PepT1. METHODS: We perfused the jejunum with a solution with or without MDP, or with a solution of MDP + Gly-Gly and explored the degree of inflammation to determine the role of PepT1-Nod2 signaling pathway in small intestine mucosa. RESULTS: MDP perfusion induced inflammatory cell accumulation and intestinal damage, accompanied by an increase in mucosal Nod2 and Rip2 transcript expression. NFκB activity and inflammatory cytokine expression, including serum levels of TNF-α, IL-1ß, and IL-6, increased in the MDP group compared to the controls; these effects were reversed by perfusion of the nutritional dipeptide Gly-Gly. CONCLUSION: MDP can be transported through PepT1, causing inflammatory damage in the rat small intestine. Nod2-Rip2-NFκB signaling involved in the small intestinal inflammatory injury caused by MDP which is transported through PepT1.

Bilateral acute pyogenic conjunctivitis with iritis induced by unilateral topical application of bacterial peptidoglycan muramyl dipeptide in adult rabbits.

The factors responsible for the conjunctivitis and iritis associated with acute ocular infection and post enteric inflammatory disease are not fully known. The pro-inflammatory activity of unilateral topical application of muramyl dipeptide (MDP; the smallest bio-active Gram-positive and Gram-negative bacterial cell wall component) was investigated in adult rabbits. The resultant bilateral conjunctivitis/iritis and pyogenic responses were characterized. Bilateral symptoms were graded by slit lamp examinations; tear fluid, Schirmer tests (tear production), blood and aqueous humor (AH) samples were obtained from MDP-treated and untreated rabbits. MDP concentration, gamma-glutamyltranspeptidase activity (GGT; key enzyme in glutathione recapture, xenobiotic detoxification, eicosanoid synthesis and neutrophil function), protein concentration, and tear cell density, cytology, and immunofluorescent antibody reactivity to GGT and calreticulin (CRT; MDP-binding protein) were determined. MDP was cleared from ipsilateral tears and serum by 6 h, but was undetected in mock-treated contralateral tears. Bilateral signs of acute transient pyogenic conjunctivitis, characterized by tearing, lid edema, conjunctival hyperemia, chemosis and leukocytic infiltrate with iritis (erythema and aqueous flare) were detected. Milder symptoms occurred in the mock-treated contralateral eyes. Bilateral symptoms, tear production, tear protein, GGT activity, and mucopurulent discharge (containing up to 2.5-5.0 × 10(6) cells/mL) were elevated 4-8 h post MDP and resolved to near pre-treatment levels by 24 h. Tear GGT activity and protein levels were higher in MDP-treated and mock-treated contralateral eyes than in eyes of untreated adult rabbits (p's < 0.001). Elevated tear GGT activity was associated with histopathology and increased vascular and epithelial permeability to serum protein, GGT-positive epithelia cells, macrophages and heterophils. Repeat MDP applications induced recurrent induction and resolution patterns of bilateral conjunctivitis/iritis and tear GGT activity, but ipsilateral GGT responses were lower. The results suggest unilateral topical MDP application to adult rabbit eyes induces a bilateral acute pyogenic conjunctivitis/iritis (PCI) characterized by increased vascular and epithelial permeability similar to acute bacterial conjunctivitis in man. The detection of CRT/GGT positive heterophils in tears suggests efferocytosis (phagocytosis of dead/dying cells). Tear GGT activity may be a useful means to quantify MDP-induced toxicity and extraocular inflammation.

Body temperature responses of Pekin ducks (Anas platyrhynchos domesticus) exposed to different pathogens.

Poultry, like mammals and other birds, develop fever when exposed to compounds from gram-negative bacteria. Mammals also develop fever when exposed to the constituents of viruses or gram-positive bacteria, and the fevers stimulated by these different pathogenic classes have discrete characteristics. It is not known whether birds develop fever when infected by viruses or gram-positive bacteria. Therefore, we injected Pekin ducks with muramyl dipeptide, the cell walls of heat-killed Staphylococcus aureus, or the viral mimic polyinosinic:polycytidylic acid and monitored their body temperature (T(b)). For comparative purposes we also injected a group of ducks with lipopolysaccharide, the only known pyrogen in birds. We then compared the T(b) invoked by each injection with the T(b) after an injection of saline. Muramyl dipeptide did not affect T(b). The cell walls of heat-killed S. aureus invoked long-lasting, dose-dependent fevers with relatively low magnitudes. Polyinosinic:polycytidylic acid invoked dose-dependent fevers with high febrile peaks. Fever is a well-known clinical sign of infection in mammals, and the results of this study indicate that the pattern of increase in T(b) could serve as an indicator for diverse pathogenic diseases in birds.

Necrotic inflammatory reaction induced by muramyl dipeptide in guinea pigs sensitized by tubercle bacilli.

In the course of studies aimed at determining whether MDP was antigenic or not, a hitherto unreported phenomenon was noticed. Injection (a provocative injection) of muramyl dipeptide (MDP) caused severe inflammation, with hemorrhage and necrosis in the footpads of guinea pigs, where tubercle bacilli in water-oil emulsion (a preparatory injection) had been injected 3-8 wk earlier. Sometimes the reaction was accompanied by generalized and fatal shock. Several related substances were tested, and only a combination of tubercle bacilli, or MDP plus proteins as the preparatory injection, and MDP as the provocative injection was found to induce this inflammatory necrotic reaction. Development of delayed hypersensitivity to protein antigens may be important for priming, but MDP and not the protein antigens provoked the reaction. This reaction was, so far, only observed in guinea pigs. Although this reaction appears to be similar to the Shwartzman reaction, the two reactions were found to differ from each other in several important points.

Activation of NOD2 in vivo induces IL-1beta production in the eye via caspase-1 but results in ocular inflammation independently of IL-1 signaling.

Nucleotide-binding and oligomerization domain 2 (NOD2) belongs to the emerging Nod-like receptor (NLR) family considered important in innate immunity. Mutations in NOD2 cause Blau syndrome, an inherited inflammation of eye, joints, and skin. Mutations in a homologous region of another NLR member, NALP3, cause autoinflammation, wherein IL-1beta plays a critical role. Here, we tested the hypothesis that IL-1beta is a downstream mediator of NOD2-dependent ocular inflammation. We used a mouse model of NOD2-dependent ocular inflammation induced by muramyl dipeptide (MDP), the minimal bacterial motif sensed by NOD2. We report that MDP-induced ocular inflammation generates IL-1beta and IL-18 within the eye in a NOD2- and caspase-1-dependent manner. Surprisingly, two critical measures of ocular inflammation, leukocyte rolling and leukocyte intravascular adherence, appear to be completely independent of IL-1 signaling effects, as caspase-1 and IL-1R1-deficient mice still developed ocular inflammation in response to MDP. In contrast to the eye, a diminished neutrophil response was observed in an in vivo model of MDP-induced peritonitis in caspase-1-deficient mice, suggesting that IL-1beta is not essential in NOD2-dependent ocular inflammation, but it is involved, in part, in systemic inflammation triggered by NOD2 activation. This disparity may be influenced by IL-1R antagonist (IL-1Ra), as we observed differential IL-1Ra levels in the eye versus plasma at baseline levels and in response to MDP treatment. This report reveals a new in vivo function of NOD2 within the eye yet importantly, distinguishes NOD2-dependent from NALP3-dependent inflammation, as ocular inflammation in mice occurred independently of IL-1beta.

Muramyl dipeptide enhances the response to endotoxin to cause multiple organ injury in the anesthetized rat.

Nucleotide oligomerization domain (NOD) proteins recognize peptidoglycan fragments, resulting in up-regulation of transcription factors, and may enhance the inflammatory response to infection. Specifically, NOD2 has been shown to sense muramyl dipeptide (MDP), which is released during bacterial cell growth and replication. Activation of NOD2 by MDP enhances the inflammatory response caused by LPS (endotoxin). Here, we investigated the effects of MDP on the organ injury/dysfunction caused by systemic administration of a low dose of LPS. Male Wistar rats were coadministered with either MDP (1 - 10 mg kg(-1), i.v.) or vehicle (0.5 mL kg(-1) saline, i.v.), and a low dose of LPS (1 mg kg(-1), i.v.) or vehicle (1 mL kg(-1), saline, i.v.). MAP and heart rate were continuously monitored for 6 h. Markers of organ dysfunction/injury, plasma cytokine levels, and lung myeloperoxidase activity were measured 6 h after MDP and LPS coadministration. In a separate study, MDP (3 or 10 mg kg(-1), i.v.) or vehicle (0.5 mL kg(-1) saline, i.v.) was administered 24 h before LPS infusion. When compared with animals receiving low-dose LPS alone, coadministration of MDP (10 mg kg(-1), i.v.) and LPS, or administration of MDP (10 mg kg(-1), i.v.) 24 h before LPS resulted in a significant increase in the degree of organ injury, cytokine release, and lung injury caused by LPS alone. Thus, our results demonstrate that the two bacterial wall components MDP and LPS work in concert to cause multiple organ injury and systemic inflammation. We hope that our results stimulate other studies designed to evaluate the effects of NOD ligands in animal models of inflammation.

Uveitis secondary to bacterial products.

Bacteria are suspected contributors to several forms of immune-mediated, noninfectious forms of uveitis including that associated with ankylosing spondylitis, sarcoidosis, Behçet's disease and inflammatory bowel disease. Endotoxin (lipopolysaccharide)-induced uveitis has been a widely used model for more than 2 decades. Both rats and mice develop a transient, bilateral anterior uveitis after a systemic injection of endotoxin. Inflammation posterior to the lens is generally milder than anterior segment inflammation. The uveitis is severer if the lipopolysaccharides are injected intraocularly. The model has been invaluable in helping to identify mediators induced in the inflamed eye and in testing pharmacologic approaches to reduce eye inflammation. Muramyl dipeptide is another bacterial cell component that can induce uveitis in laboratory animals. Muramyl dipeptide is especially intriguing as a cause of uveitis because it activates the intracellular protein, Nod2, and mutations in the NOD2 gene are the cause of the autosomal dominant form of uveitis that is characteristic of Blau syndrome. Since a mutation in a gene that codes for a protein which senses a bacterial product consistently results in uveitis, it is critical to understand more fully the mechanisms by which bacterial products cause uveitis in laboratory animals.

MDP and other muropeptides--direct and synergistic effects on the immune system.

Muropeptides are breakdown products of peptidoglycan (PGN) of Gram-negative and Gram-positive bacteria. They are released during bacterial growth and division, as part of the host response by lysozyme and amidases, or upon antibiotic treatment. After phagocytosis of bacteria or bacterial breakdown products by host immune cells, the muropeptides trigger intracellular signaling cascades, leading to altered gene expression and activation of the immune response. Numerous muropeptides and derivatives have been synthesized chemically to characterize their immunostimulatory effects and adjuvant activity. Muramyl dipeptide, a natural partial structure of PGN, is the minimal structure with adjuvant activity. This review discusses the structure and occurrence of muropeptides and gives a broad overview of their inflammatory and adjuvant activity and the possible involvement of receptors in these responses.

Muramyl peptides augment cytotoxic effect of tumor necrosis factor-alpha in combination with cytotoxic drugs on tumor cells.

We have demonstrated that biologically active muramyl peptides, in particular, glucosaminylmuramyl dipeptide (GMDP), augmented in vitro cytotoxic activity of tumor necrosis factor-alpha (TNF-alpha) against murine fibrosarcoma L929 cells. The introduction of GMDP resulted in cytotoxic effect characteristic for substantially higher dose of cytokine. Even more potent was the combination of GMDP, TNF-alpha and Actinomycin D (ActD). According to clonogenic and MTT assays 100% L929 cells could be killed in culture with low doses of TNF-alpha and ActD if GMDP was present. When cisplatin was substituted for ActD similar results were obtained. GMDP also enhanced cytotoxicity of TNF-alpha and cisplatin against human breast carcinoma MCF7 and histiocytic lymphoma U937 cells. Normal cells, namely human peripheral blood leucocytes and murine peritoneal macrophages, were resistant to selected doses of TNF-alpha/cisplatin/GMDP.

Enhancement of endotoxic shock by N-acetylmuramyl-L-alanyl-(L-seryl)-D-isoglutamine (muramyl dipeptide).

We described elsewhere that the synergistic antitumor activity of endotoxic extracts from Re mutants of gram-negative bacteria and trehalose mycolate against guinea pig syngeneic line 10 tumor was abrogated after peptide substances accompanying these extracts had been removed. This activity could be restored by combining peptide-free endotoxin either with cell wall skeleton from Bacillus Calmette-Guérin, a polymeric mycolic acid-arabinogalactan-mucopeptide complex, or with a combination of two separate components, trehalose dimycolate and N-acetylmuramyl-L-alanyl-(L-seryl)-D-isoglutamine (MDP). We report here that when a combination of endotoxin (150 microgram) and a mixture of MDP (150 microgram) and trehalose dimycolate (150 microgram) was inoculated into established dermal tumors, a significant number of the animals died, presumably of endotoxic shock. All surviving animals suffered severe but temporary lethargy. When administered alone intradermally in the dose levels tested, none of the components caused severe lethargy or lethality. The lethal effects of 159 microgram of MDP also occurred in combination with relatively weak endotoxic products, such as Pseudomonas vaccine (Pseudogen), and these effects did not depend upon the presence of malignant tissue. Guinea pigs inoculated i.v. were even more susceptible inasmuch as the addition of as little as 6 microgram of MDP to 150 microgram of Pseudogen, itself not lethal, caused the death of 80% of the animals.

Phase I and immunomodulatory study of a muramyl peptide, muramyl tripeptide phosphatidylethanolamine.

Muramyl tripeptide phosphatidylethanolamine (MTP-PE; CGP 19835A from Ciba Geigy) is a synthetic muramyl tripeptide structurally related to bacterial cell wall constituents. MTP-PE activates monocytes in vitro to a tumoricidal state and has in vivo antitumor effects in animal models. We studied the toxicity and immunomodulatory effects of once weekly i.v. administration of liposomal-encapsulated MTP-PE for 8 weeks in 27 patients with advanced malignancies. Doses ranged from 0.1 to 2.7 mg/m2. No major tumor responses were seen; 11 patients had stable disease after 8 weeks of therapy and 3 continued on maintenance therapy because of minor tumor regressions and/or clinical improvement. MTP-PE at these doses was well tolerated. Shaking chills and fevers were the most common toxicities and occurred at all dose levels. There was no treatment-induced loss of performance status. Immunomodulatory studies revealed evidence of a biological effect on monocytes. C-reactive protein levels rose in the majority of patients with end-of-treatment values 2 to 10 times higher than baseline. Serum neopterin levels were consistently increased 24 h after MTP-PE administration and significant decreases in expression of two different types of Fc receptors on peripheral blood monocytes were noted 6 h after treatment. Although no major tumor responses were seen in this group of patients with advanced malignancies, MTP-PE was well tolerated and exerted biological effects on monocytes. Serum neopterin levels may be a useful marker for the biological effects of MTP-PE.

Liposomal phosphatidylserine inhibits tumor cytotoxicity of liver macrophages induced by muramyl dipeptide and lipopolysaccharide.

Liposomes can very efficiently deliver immunomodulators to macrophages so as to induce tumor cytotoxicity. Liposomes most widely used for that purpose contain negatively charged lipids, in particular phosphatidylserine (PS), to enhance liposome uptake by the macrophages. We investigated the effect of three negatively charged liposomal lipids on the in vitro activation of liver macrophages to tumor cytotoxicity by muramyl dipeptide (MDP) and lipopolysaccharide (LPS). Both MDP- and LPS-induced tumor cytotoxicity towards murine colon adenocarcinoma cells were strongly inhibited by PS-containing liposomes. Under comparable conditions phosphatidylglycerol (DPPG)-containing or dicetyl phosphate (DCP)-containing liposomes did not inhibit or only marginally inhibited the induction of tumor cytotoxicity. We did not observe PS-mediated inhibition of tumor cell toxicity when the exposure of the macrophages to PS-liposomes was limited to the 4-h activation period prior to addition of the tumor target cells, suggesting that the inhibitory effect is accomplished at the level of the later stages of the activation process. Previously, we showed that macrophages which are activated to tumor cytotoxicity during a 24-h incubation with MDP become refractory to a second activation with MDP. Now we observed that simultaneous incubation with PS-containing liposomes partially prevents this refractoriness, which is also compatible with an interfering action of PS at a relatively late stage in the activation process. We conclude that PS, despite its reported stimulatory effect on liposome uptake by macrophages, can seriously antagonize the effectiveness of immunomodulating agents acting on macrophages. This bears relevance to the use of PS-containing liposomes as a vehicle for such agents. The results are discussed in perspective of earlier reported pharmacological effects of PS and its metabolites.

Changes in rabbit febrile responses to muramyl dipeptide (MDP) after coupling to a synthetic carrier.

Muramyl dipeptide (MDP) is a small molecular weight synthetic glycopeptide (less than 500), which has been shown to be an immunoadjuvant, and to induce a biphasic febrile response in the rabbit--probably via the release of endogenous pyrogen--accompanied by a marked leukopenia. Macromolecularization by coupling to a synthetic carrier (MW approximately or equal to 60,000) potentiates the immunostimulant properties of MDP but also its pyrogenicity. The present study demonstrates that such a conjugate induced the release of endogenous pyrogen in vivo and in vitro at lower dosage levels than free MDP. Further experiments showed that there existed several differences between free and conjugated MDP. Thus, after intravenous administration of the conjugate, the fever pattern was monophasic with a prompt defervescence and not accompanied by leukopenia at dosage levels inducing similar increase in body temperature. In addition, when fever was recorded after intracerebroventricular administration, the increase in sensitivity was much greater in the case of free MDP than of MDP-A--L.

Priming effect of muramyl peptides for induction by lipopolysaccharide of tumor necrosis factor production in mice.

Lipopolysaccharide-induced necrosis of grafted tumors was potentiated by several hydrophilic and lipophilic muramyl dipeptide (MDP) derivatives administered a few hours prior to small amounts of lipopolysaccharide (LPS) in spite of low titers of induced circulating tumor necrosis factor (TNF). However, pretreatment with MDP derivatives did increase the level of TNF in the blood of mice challenged by a greater dose of LPS. The TNF amount in 2 h postendotoxin mouse serum reached a peak when the glycopeptide had been given 6 h before the challenge, being approximately 100-fold above that obtained in unprimed mice. The cytotoxic activity in mouse serum was inhibited by rabbit antibodies raised against recombinant mouse TNF. Although there exists a toxic synergism between BCG or MDP and endotoxin, the effect of certain MDP derivatives was not related to an increased susceptibility to the toxicity of LPS.

Human monocytoid cell lines as indicators of endotoxin: comparison with rabbit pyrogen and Limulus amoebocyte lysate assay.

The aim of this study was to develop an in vitro test system for pyrogenic substances. Three clones derived from human monocytoid cell lines, which were selected by their high sensitivity to lipopolysaccharide (LPS), were assessed for tumor necrosis factor (TNF) production. Their response to pyrogen-containing samples was compared with that in a Limulus amoebocyte lysate assay and the rabbit pyrogen test. We show here that the induction of TNF in these clones is a valid in vitro alternative to determine endotoxin in commercial preparations requiring pyrogenicity testing. Cell clones derived from Mono Mac 6 (MM6 2H8 and MM6 4B5) responded to sub-ng/ml concentrations of complete rough-strain and smooth-strain LPS, to ng/ml concentrations of diphosphoryl-lipid A, and to microgram/ml concentrations of monophosphoryl-lipid A and to detoxified LPS. Cells reacted to > or = 1 microgram/ml lipoteichoic acid by TNF production, and were relatively insensitive to toxic shock syndrome toxin-1 (TSST-1) and to muramyl dipeptide adjuvant peptide. The reaction pattern of a clone derived from THP-1 (THP-1 1G3) was in general, similar to that of the MM6 clones, except that THP-1 1G3 failed to react to diphosphoryl-lipid A. When tested on commercial samples destined for parenteral use, there was a close correlation between a sensitive Limulus amoebocyte lysate (LAL) test and the cell culture test on the one hand, and between the pyrogen test and the cell culture test on the other hand. The data suggest that this cell-based test is able to recognize pyrogens derived from gram-negative organisms in test samples with appropriate sensitivity and specificity. This test appears to be able to eliminate some of the false-positive data obtained in the LAL test.

1998 Curt P. Richter Award. Brain mechanisms in cytokine-induced anorexia.

Our research focuses on the mechanisms underlying cytokine action in the central nervous system (CNS) using an integrative and multidisciplinary strategy organized through supracellular (behavioral analysis by computerized monitoring systems), cellular (extracellular and intracellular neurophysiological recording), and molecular (patch-clamp recording, and DNA, RNA and protein analyses) approaches. An integrative strategy that combines computerized meal pattern analyses with cellular and molecular biology approaches allows the study of underlying brain mechanisms in cytokine- and disease-associated anorexia. This paper presents a comprehensive discussion of our laboratory's previously published data on brain mechanisms involved in cytokine-induced anorexia including the relevance of meal pattern analysis (meal size, meal duration, meal frequency, intermeal intervals), modulation of hypothalamic neuronal activity, molecular processes involving ionic conductances, cytokine-cytokine and cytokine-peptide interactions, and modulation of cytokine and peptide/neuropeptide system components (ligands, endogenous inhibitors, receptor subtypes, signal transduction molecules, intracellular mediators) and cytokine feedback systems.

Tolerance to pyrogens.

In humans or experimental animals, the repeated confrontation with lipopolysaccharides (LPS) from gram-negative bacteria, but not with muramyl dipeptide (MDP) from gram-positive bacteria, leads to attenuation of almost all pathophysiologic effects mediated by proinflammatory cytokines. Our experiments in guinea pigs and rats demonstrate that attenuation of the febrile response during the development of LPS tolerance is associated with a reduced production of cytokines rather than a decrease in responsiveness to cytokines. Cross-tolerance experiments demonstrate that different stimuli influencing LPS-induced tumor necrosis factor (TNF) release and nitric oxide (NO) synthesis can modify the development of tolerance. On the other hand, the lack of cross-tolerance between LPS and MDP indicates that MDP can activate the cytokine cascade and induce the febrile response in animals tolerant to LPS. This may indicate distinct receptors and signal pathways for LPS and MDP, leading to activation of the cytokine cascade. LPS tolerance has also been demonstrated in ex vivo and in vitro studies. In cultures of monocytes, diminished synthesis of TNF and NO reported after LPS restimulation could be prevented and reversed by interferon and granulocyte-macrophage colony-stimulating factor. These findings add an additional hypothesis in tolerance development.

The effect of alpha-MSH on fever caused by Staphylococcus aureus cell walls in rabbits.

The role of endogenous pyrogens induced by gram-positive bacterial pyrogens is not known. Intravenous alpha-MSH (2.5 micrograms) significantly reduced only the first phase of the biphasic thermal response to IV S. aureus cell walls (5 x 10(7)). Intracerebroventricular alpha-MSH (200 ng) had no effect on the fever response. The fall in serum iron concentration was significantly attenuated by the IV alpha-MSH but was not affected by the ICV alpha-MSH. Intravenous alpha-MSH had no effect on fever or the serum iron response caused by muramyl dipeptide (MDP). We conclude that the first phase of the thermal response to S. aureus cell walls is mediated by an endogenous pyrogen (EP) and the second phase of the response by a mechanism not involving EP, but possibly a muramyl peptide.

Pharmacological aspects of arthritis induced by a muramyl dipeptide analogue in rats.

Fourteen consecutive daily subcutaneous injections of 4 mg/kg of the muramyl dipeptide analogue MDP-Lys(L18) into rats caused arthritis characterized by swelling of the tarsal joint, increases in lymphocytes and monocytes in the peripheral blood, and elevated serum immunoglobulin G (IgG). The present study was performed to evaluate the effects of indomethacin, phenylbutazone, dexamethasone, D-penicillamine, aurothioglucose, cyclophosphamide and cyclosporin A on this arthritis. Administration of indomethacin, phenylbutazone or dexamethasone inhibited the development of the tarsal joint swelling, suggesting that prostaglandins may be involved in the pathogenesis of the arthritis. Cyclophosphamide reduced the arthritis, together with decreases in the lymphocyte count and the serum IgG level. Cyclosporin A worsened the arthritis in a dose-dependent manner and increased the neutrophil count without raising the serum IgG level, but inhibited the induction of adjuvant arthritis in rats with Mycobacterium bacilli. MDP-Lys(L18) may therefore induce arthritis differing in mechanism from adjuvant arthritis.