Kouji Yamaguchi

Enhancement by galactosamine of lipopolysaccharide(LPS)-induced tumour necrosis factor production and lethality: its suppression by LPS pretreatment

D‐Galactosamine (GalN) depletes UTP primarily in the liver, resulting in decreased RNA synthesis in hepatocytes. Co‐injection of GalN and lipopolysaccharide (LPS) into mice produces fulminant hepatitis with severe hepatic congestion, resulting in rapid death. Although the underlying mechanism is uncertain, GalN enhances the sensitivity to tumour necrosis factor (TNF). Administration of uridine (a precursor of UTP) prior injection of either LPS itself or interleukin‐1 (IL‐1) reduces the lethality of GalN+LPS. The present study focused on the effects of these agents on TNF production. Intraperitoneal injection of GalN+LPS into mice greatly elevated serum TNF. Although large doses of LPS alone also greatly elevated serum TNF, LPS itself induced neither hepatic congestion nor rapid death. Administration of a macrophage depletor, liposomes encapsulated with dichloromethylene bisphosphonate, reduced both the TNF production and mortality induced by GalN+LPS. Uridine, when injected 0.5 h after the injection of GalN+LPS, reduced the production of TNF. Prior injection of LPS, but not of IL‐1, also reduced this TNF production. Serum from LPS‐injected mice reduced the TNF production induced by GalN+LPS, but it was less effective at reducing the lethality. Its ability to reduce TNF production was abolished by heat‐treatment. We hypothesize that a factor inhibiting TNF production by macrophages is produced by hepatocytes in response to LPS. Possibly, production of this hepatocyte‐derived TNF‐down‐regulator (TNF‐DRh) may be: (i) inhibited by GalN, causing over‐production of TNF by macrophages and (ii) stimulated by LPS‐pretreatment (and restored by uridine), causing reduced TNF production.

Involvement of interleukin-1 in the inflammatory actions of aminobisphosphonates in mice

Aminobisphosphonates (aminoBPs) are potent inhibitors of bone resorption. However, they cause undesirable inflammatory reactions, including fever, in humans. Intraperitoneal injection of aminoBPs into mice also induces inflammatory reactions, including a prolonged elevation of the activity of the histamine‐forming enzyme, histidine decarboxylase (HDC). Because interleukin‐1 (IL‐1) is a typical pyrogen and a strong inducer of HDC, we examined whether aminoBPs induce inflammatory reactions in mice deficient in genes for both IL‐1α and IL‐1β (IL‐1‐KO mice). In control mice, aminoBPs induced an elevation of HDC activity and other inflammatory reactions (enlargement of the spleen, atrophy of the thymus, exudate in the thorax and increase in granulocytic cells in the peritoneal cavity). These responses were all weak or undetectable in IL‐1‐KO mice. We have previously shown that lipopolysaccharides (LPSs) from Escherichia coli and Prevotella intermedia (a prevalent gram‐negative bacterium both in periodontitis and endodontal infections) are capable of inducing HDC activity in various tissues in mice. In control mice treated with an aminoBP, the LPS‐induced elevations of serum IL‐1 (α and β) and tissue HDC activity were both markedly augmented. However, such an augmentation of HDC activity was small or undetectable in IL‐1‐KO mice. These results, taken together with our previous findings (i) suggest that IL‐1 is involved in the aminoBP‐induced inflammatory reactions and (ii) lead us to think that under some conditions, inflammatory reactions induced by gram‐negative bacteria might be augmented in patients treated with an aminoBP. In this study, we also obtained a result suggesting that IL‐1‐deficiency might be compensated by a second, unidentified, mechanism serving to induce HDC in response to LPS when IL‐1 is lacking.

Inhibition of inflammatory actions of aminobisphosphonates by dichloromethylene bisphosphonate, a non‐aminobisphosphonate

When injected intraperitoneally into mice in doses larger than those used clinically, all the amino derivatives of bisphosphonates (aminoBPs) tested induce a variety of inflammatory reactions such as induction of histidine decarboxylase (HDC, the histamine‐forming enzyme), hypertrophy of the spleen, atrophy of the thymus, hypoglycaemia, ascites and accumulation of exudate in the thorax, and an increase in the number of macrophages and/or granulocytes in the peritoneal cavity of blood. On the other hand, dichloromethylene bisphosphonate (Cl2MBP) a typical non‐aminoBP, has no such inflammatory actions. In the present study, we found that this agent can suppress the inflammatory actions of aminoBPs. Cl2MBP, when injected into mice before or after injection of 4‐amino‐1‐hydroxybutylidene‐1,1‐bisphosphonic acid (AHBuBP; a typical aminoBP), inhibited the induction of HDC activity by AHBuBP in a dose‐ and time‐dependent manner. The increase in HDC activity induced by AHBuBP was largely suppressed by the injection of an equimolar dose of Cl2MBP. Cl2MBP also inhibited other AHBuBP‐induced inflammatory reactions, as well as the inflammatory actions of two other aminoBPs. However, Cl2MBP did not inhibit the increase in HDC activity induced by lipopolysaccharide (LPS). We have previously reported that AHBuBP augments the elevation of HDC activity and the production of interleukin‐1β (IL‐1β) that are induced by LPS. These actions of AHBuBP were also inhibited by Cl2MBP. Based on these results and reported actions of bisphosphonates, the mechanisms underlying the contrasting effects of aminoBPs and Cl2MBP, a non‐aminoBP are discussed. The results suggest that combined administration of Cl2MBP and an aminoBP in patients might be a useful way of suppressing the inflammatory side effects of aminoBPs.

Inhibition of necrotic actions of nitrogen-containing bisphosphonates (NBPs) and their elimination from bone by etidronate (a non-NBP): a proposal for possible utilization of etidronate as a substitution drug for NBPs.

PURPOSE Nitrogen-containing bisphosphonates (NBPs) have powerful anti-bone-resorptive effects (ABREs). However, recent clinical applications have disclosed an unexpected side effect, osteonecrosis of the jaw. We previously found in mice that etidronate (a non-NBP), when coadministered with alendronate (an NBP), inhibited the latters inflammatory effects. However, etidronate also reduced the ABRE of alendronate. The present study examined in mice the modulating effects of etidronate on the inflammatory and necrotic actions of zoledronate (the NBP with the strongest anti-bone-resorptive activity and the highest incidence of osteonecrosis of the jaw) and on ABREs of various NBPs including zoledronate. MATERIALS AND METHODS NBPs were subcutaneously injected into ear pinnas of mice and ensuing inflammation and necrosis at the site of the injection were evaluated. ABREs of NBPs were evaluated by analyzing sclerotic bands induced in mouse tibias. RESULTS Coinjection of etidronate reduced inflammatory and necrotic reactions induced by zoledronate, and also reduced the amount of zoledronate retained within the ear tissue. When both agents were intraperitoneally injected, etidronate reduced the ABRE of zoledronate and those of other NBPs. Notably, etidronate reduced the ABRE of zoledronate even when this non-NBP was injected 16 hours after the injection of zoledronate. Bone scintigram indicated that etidronate reduced the amount of zoledronate that had already bound to bone. CONCLUSIONS These results suggest that etidronate may 1) inhibit the entry of NBPs into cells related to inflammation and/or necrosis, 2) inhibit the binding of NBPs to bone hydroxyapatite, 3) at least partly eliminate (or substitute for) NBPs that have already accumulated within bones, and thus 4) if used as a substitution drug for NBPs, be effective at treating or preventing NBP-associated osteonecrosis of the jaw.

Necrotic actions of nitrogen-containing bisphosphonates and their inhibition by clodronate, a non-nitrogen-containing bisphosphonate in mice: potential for utilization of clodronate as a combination drug with a nitrogen-containing bisphosphonate.

Nitrogen-containing bisphosphonates (NBPs) exhibit powerful anti-bone-resorptive effects (ABREs) via inhibition of farnesyl pyrophosphate synthase during cholesterol biosynthesis. Clinical applications have disclosed an unexpected side effect, namely osteonecrosis of jaw bones, and although thousands of cases have been documented in the last few years the mechanism remains unclear. Since NBPs accumulate in bone-hydroxyapatite, more jaw bone osteonecrosis cases may come to light if NBPs continue to be used as they are being used now. We have previously reported that in mice, systemic (intraperitoneal) injection of clodronate (a non-NBP) prevents the inflammatory effects of NBPs. Here, we examined in mice the local necrotic actions of various NBPs and the anti-necrotic effects of clodronate. A single subcutaneous injection of an NBP into the ear pinna induced necrosis at the injection site (relative potencies of necrotic actions of NBPs: zoledronate >> pamidronate > or = alendronate > risedronate), while non-NBPs lacked this effect. Clodronate, when injected together with an NBP, reduced or prevented the necrosis induced by that NBP, but not its ABRE. Clodronate reduced the amount of each NBP retained within tissues. These results, together with those of previous studies, suggest that (i) clodronate inhibits the inflammatory and necrotic actions of NBPs by inhibiting their incorporation into cells related to inflammation and/or necrosis, (ii) clodronate could be useful as a combination drug with NBPs for preventing their necrotic actions while retaining their ABREs and (iii) clodronate could also be useful as a substitution drug for NBPs in patients at risk of osteonecrosis of jaw bones.

Induction of histidine decarboxylase, the histamine-forming enzyme, in mice by interleukin-12

Interleukin (IL)-12, a potent antitumour cytokine, has inflammatory side effects. We examined the effect of IL-12 on the histamine-forming enzyme, histidine decarboxylase (HDC). When injected intraperitoneally into C3H/HeN mice, IL-12 exhibited antitumour activity against squamous epithelial tumour cells (NR-S1 cells). At doses that produced this antitumour activity, IL-12 also enhanced HDC activity in the lung, liver, spleen and bone marrow. Compared with that induced by IL-1, the elevation of HDC activity induced by IL-12 was low and slow. However, daily injections of IL-12, but not of IL-1, produced a cumulative effect on HDC activities, an accumulation of exudate in the thorax, and death. Antagonists of H1 and H2 receptors and an inhibitor of HDC all failed to prevent the pulmonary exudation and death. These results suggest that IL-12 is an inflammatory cytokine capable of stimulating the synthesis of histamine, but that histamine itself may be not the direct cause of the pulmonary exudation and/or lethality induced by IL-12.

Elevation of histidine decarboxylase activity in the stomach of mice by ulcerogenic drugs

Histamine is involved in the development of gastric lesions. To examine the contribution of the histamine-forming enzyme, histidine decarboxylase, to drug-induced gastric lesions, we compared the effects of aspirin, indomethacin and dexamethasone on histidine decarboxylase activity in mice. Administration of these drugs, orally or intraperitoneally, elevated histidine decarboxylase activity in the stomach but not in the liver, lung or spleen, dexamethasone being the most potent. In contrast, acetaminophen (a non-ulcerogenic drug) was inactive. These results and our previously reported findings (elevation of histidine decarboxylase activity by lipopolysaccharide, interleukin-1 and tumour necrosis factor, and by different types of stress) suggest that an elevation of histidine decarboxylase activity in the stomach may be a common feature of the responses to ulcerogenic stimuli. The possible participation of histidine decarboxylase in gastric lesions is discussed on the basis of the known actions of histamine, our findings and the effect of histamine H(2) receptor antagonists on histidine decarboxylase activity.

Roles of platelets and macrophages in the protective effects of lipopolysaccharide against concanavalin A-induced murine hepatitis.

Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment+ConA induced hepatitis. In mice deficient in IL-1α and IL-1β (but not in TNFα), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types.

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.

A Strategy against the Osteonecrosis of the Jaw Associated with Nitrogen-Containing Bisphosphonates (N-BPs): Attempts to Replace N-BPs with the Non-N-BP Etidronate

Bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ) can occur when enhanced bone-resorptive diseases are treated with nitrogen-containing BPs (N-BPs). Having previously found, in mice, that the non-N-BP etidronate can (i) reduce the inflammatory/necrotic effects of N-BPs by inhibiting their intracellular entry and (ii) antagonize the binding of N-BPs to bone hydroxyapatite, we hypothesized that etidronate-replacement therapy (Eti-RT) might be useful for patients with, or at risk of, BRONJ. In the present study we examined this hypothesis. In each of 25 patients receiving N-BP treatment, the N-BP was discontinued when BRONJ was suspected and/or diagnosed. After consultation with the physician-in-charge and with the patients informed consent, Eti-RT was instituted in one group according to its standard oral prescription. We retrospectively compared this Eti-RT group (11 patients) with a non-Eti-RT group (14 patients). The Eti-RT group (6 oral N-BP patients and 5 intravenous N-BP patients) and the non-Eti-RT group (5 oral N-BP patients and 9 intravenous N-BP patients) were all stage 2-3 BRONJ. Both in oral and intravenous N-BP patients (particularly in the former patients), Eti-RT promoted or tended to promote the separation and removal of sequestra and thereby promoted the recovery of soft-tissues, allowing them to cover the exposed jawbone. These results suggest that Eti-RT may be an effective choice for BRONJ caused by either oral or intravenous N-BPs and for BRONJ prevention, while retaining a level of anti-bone-resorption. Eti-RT may also be effective at preventing BRONJ in N-BP-treated patients at risk of BRONJ. However, prospective trials are still required.