Prime role of bone IL-1 in mice may lie in emergency Ca(2+)-supply to soft tissues, not in bone-remodeling.

IL-1 and TNF-α are thought to be important bone-remodeling regulators. However, mice lacking either them or their receptors reportedly grow healthily. Here, we examined the roles of IL-1 and TNF-α in bone. Although a significant IL-1 level was detected in the tibia of non-stimulated wild-type (WT) mice, no significant physicochemical, morphological, or histological defects were detected in the tibias in mice lacking IL-1 (both α and ß types) (IL-1KO) or lacking both IL-1 and TNF-α (IL-1/TNF-αKO). Injection of sub-lethal doses of lipopolysaccharide (LPS) into WT mice induced a transient hypocalcemia, increased IL-1 (in the plasma and markedly in the tibia), and increased TNF-α (markedly in the plasma, but only slightly in the tibia). LPS-induced hypocalcemia was modest in IL-1KO mice, and not detected in IL-1/TNFαKO mice. IL-1α (but not TNFα) induced hypocalcemia in both WT and IL-1KO mice. In both WT and IL-1KO mice treated with clodronate (osteoclast inhibitor), the LPS-induced hypocalcemia was markedly augmented. Nifedipine (inhibitor of both voltage-activated and capacitative Ca(2+)-entry) reduced the LPS-induced hypocalcemia. These results suggest that in mice: (i) IL-1 and TNF-α may contribute little to physiological bone-formation, and (ii) a time-lag between IL-1- and TNF-α-stimulated Ca(2+)-entry into cells throughout the body from the circulation and IL-1-stimulated Ca(2+)-release from the bone may cause the observed transient LPS-induced hypocalcemia. Thus, the prime role of bone IL-1 may reside in the supply of Ca(2+) from the bone to cells throughout the body when the need is urgent.

Pharmacological characterization of anaphylaxis-like shock responses induced in mice by mannan and lipopolysaccharide.

Intravenous injection of lipopolysaccharide (LPS, a component of the Gram-negative bacterial cell-surface) or mannan (Man, a component of the fungal cell-surface) into mice reportedly induces anaphylaxis-like shock (ALS) via complement-associated platelet degradation and platelet-activating factor (PAF), respectively. However, it is unclear whether PAF is involved in LPS-ALS or whether complements and/or platelets are involved in Man-ALS. Here, using preparations of Man from Saccharomyces cerevisiae and LPS from Klebsiella O3, we characterized and compared LPS-ALS and Man-ALS, with the following results. (1) ALS depended on mouse strain (ddY and BALB/c being highly responsive to Man and LPS, respectively), but not on Toll-like receptors 2 and 4. (2) In ddY mice, Man had little effect on platelets, K76 (C5a-inhibitor) did not prevent Man-ALS, and Man-ALS was augmented by prior platelet depletion. (3) CV-3988 (PAF antagonist) prevented Man-ALS, but not LPS-ALS. (4) LPS-ALS and Man-ALS were each augmented by prior injection of a muramyl dipeptide (MDP, a constituent abundant in the Gram-positive bacterial cell-surface), but prevented by prior macrophage depletion. (5) Co-administration of Man and LPS induced an augmented ALS in both ddY and BALB/c mice. These results indicate that (i) Man and LPS each induces ALS in mice in strain-dependent and macrophage-dependent (but not TLR-dependent) ways by stimulating a platelet-non-associated PAF pathway and a platelet-associated complement pathway, respectively, and (ii) these pathways are primed by MDP and exhibit mutually augmenting actions. Man-ALS and LPS-ALS may therefore serve as models for diseases involving augmentation by multiple or mixed infections.

Hepatic platelet accumulation in Fas-mediated hepatitis in mice.

Platelets are reported to be causally involved in experimental hepatitis. Jo2, an agonistic anti-Fas antibody, induces hepatitis in mice. We examined the in vivo behaviors of platelets in mice injected with this antibody (analyzed by measuring 5-hydroxytryptamine, a constituent of platelets). We found that Jo2 induces platelet accumulation predominantly in the liver, and that this hepatic platelet accumulation (HPA) precedes the increases in hepatitis markers (alanine- and asparagine-aminotransferases [ALT and AST]). By electron microscopy, we detected entry of platelets into hepatocytes, and also evidence of apoptosis among hepatocytes. A caspases-3/6/7/8/10 inhibitor prevented the Jo2-induced HPA and hepatitis. In platelet-depleted mice, contrary to our expectations, the Jo2-induced hepatitis was not reduced, and actually the increase in AST was significantly augmented, although the survival time of mice given a lethal dose of Jo2 was significantly increased (nearly doubled). Interestingly, prior induction of HPA by a low dose of lipopolysaccharide markedly reduced Jo2-induced hepatitis. Jo2 also induced HPA and hepatitis in mice deficient in both IL-1 and TNFalpha, although Jo2 increased the blood level of TNFalpha in wild-type mice. These results suggest that in Jo2-induced hepatitis: (i) platelets accumulate predominantly in the liver as a result of hepatic lesions, and that this precedes the release of transaminases from hepatocytes, and (ii) IL-1 and TNFalpha are not essential for Jo2-hepatitis. We hypothesize that platelet accumulation in the liver may, contrary to our expectations, be protective when the hepatitis is local or not severe, but harmful when hepatitis is severe.

Critical roles of platelets in lipopolysaccharide-induced lethality: effects of glycyrrhizin and possible strategy for acute respiratory distress syndrome.

Within a few minutes of an intravenous injection of a lipopolysaccharide (LPS) into mice, platelets accumulate, largely in the lung. At higher doses, LPS induces rapid shock (within 10 min), leading to death within 1 h. This type of shock differs from so-called endotoxin shock, in which shock signs and death occur several hours or more later. Here, we found that platelet depletion (by a monoclonal anti-platelet antibody) prevented LPS-induced rapid shock, but increased delayed lethality. In Japan, glycyrrhizin (GL), a compound isolated from licorice, is daily and slowly infused intravenously into chronic hepatitis C patients. A single bolus intravenous injection into mice of GL (200 mg/kg or less) shortly before (or simultaneously with) LPS injection reduced the pulmonary platelet accumulation and the severity of the rapid shock, and prevented death in both the early and later periods. GL itself, at 400 mg/kg, produced no detectable abnormalities in the appearance or activity of mice. Intraperitoneal injection of aspirin or dexamethasone had only marginal effects on LPS-induced platelet responses and lethality. These results suggest that platelets play important roles in the development of both the rapid and delayed types of shock induced by LPS. Although the mechanism by which GL suppresses platelet responses and delayed lethality remains to be clarified, GL might provide a strategy for alleviating the acute respiratory distress syndrome seen in sepsis. Our results may also support the proposal by Cinatl et al. [Cinatl J, Morgenstern B, Bauer G, Chandra P, Ravenau H, Doerr HW. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003; 361: 2045-6.] that GL may be an effective drug against severe acute respiratory syndrome.

LPS-induced platelet response and rapid shock in mice: contribution of O-antigen region of LPS and involvement of the lectin pathway of the complement system.

Intravenous injection of a lipopolysaccharide (LPS) into mice induces a rapid accumulation of platelets in the lung and liver. When degradation of the accumulated platelets occurs, anaphylactoid shock follows rapidly, the severity of the shock paralleling the quantity of platelets accumulated in the lung. Here we examined the contributions made by LPS structure and the complement system to the platelet response to LPS. BALB/c mice were injected with an LPS from Escherichia coli O8, O9, O111, or K-12, or from recombinant mutants of K-12. The O-regions of the O8 and O9 LPSs consist of a mannose homopolysaccharide (MHP), while that of O111 consists of a heteropolysaccharide (not including mannose), and K-12 LPS lacks an O-region. O111 LPS was devoid of the ability to induce the platelet response or shock, while the ability of K-12 LPS was weak. The 2 recombinant LPSs-each having an O-region (from O8 or O9) linked to K-12 LPS-exhibited activities similar to or stronger than those of their original LPSs. Mannose-binding lectin (MBL) complexed with MBL-associated serine proteases (MASPs) bound strongly to LPSs containing MHP and caused C4 activation. Moreover, the abilities of these LPSs to activate the complement system corresponded well with their abilities to induce the platelet response and rapid shock. These results suggest that the structure of the O-antigen region is important for the platelet response to LPS, and that activation of the lectin pathway of the complement system is involved in this response.