Ruth Ann Henriksen

Splenic PGE2-releasing macrophages regulate Th1 and Th2 immune responses in mice treated with heat-killed BCG

Hosts infected with low doses of mycobacteria develop T helper cell type 1 (Th1) immunity, but at relatively higher doses, a switch to Th2 immunity occurs. Prostaglandin E2 (PGE2) is a proposed mediator of the Th1‐to‐Th2 shift of immune responses, and mycobacterial products induce PGE2‐releasing macrophages (PGE2‐MØ) in the mouse spleen in a dose‐dependent manner. Splenic PGE2‐M Ø from Balb/c mice, given 0.01 or 1 mg heat‐killed (HK) Mycobacterium bovis bacillus Calmette‐Guerin (BCG) intraperitoneally (i.p.), were characterized by the ex vivo release of PGE2 (>10 ng/106 cells), cytokine production, and expression of PGG/H synthase (PGHS)‐1, PGHS‐2, cytosolic PGE synthase (PGES), and microsomal PGES‐1. At Day 14 after the treatment, mice treated with 1 mg, but not 0.01 mg, BCG had increased levels of PGHS‐2+ PGE2‐MØ, total serum immunoglobulin E (IgE), and serum IgG1 antibodies (Th2 responses) against heat shock protein 65 and purified protein derivative. Cultures of spleen cells isolated from these mice expressed interleukin (IL)‐4 and IL‐10 in recall responses. Treatment of mice receiving 1 mg BCG with NS‐398 (a PGHS‐2 inhibitor, 10 mg/kg i.p., daily) resulted in enhanced interferon‐γ (IFN‐γ) production with reduced IL‐4 and IL‐10 production in recall responses. This treatment also resulted in decreased total serum IgE levels. Treatment of C57Bl/6 mice with HK‐BCG (0.5 mg dose) also induced a mixture of Th1 and Th2 responses, although IFN‐γ production was markedly increased, and IL‐4 was decreased compared with Balb/c mice. Thus, our results indicate that by 14 days following treatment of mice with high doses of HK‐BCG, splenic PGE2‐MØ formation is associated with a PGHS‐2‐dependent shift from Th1‐to‐Th2 immune responses.

Cardiac and Vasular Changes in Mice After Exposure to Ultrafine Particulate Matter

Increased ambient air particulate matter (PM) concentrations are associated with risk for myocardial infarction, stroke, and arrhythmia, and ultrafine PM (UFPM) might be particularly toxic to the cardiovascular system. Recent epidemiological studies are beginning to offer mechanistic insights, yet the rodent model remains a valuable tool to explore potential mechanisms. This article reviews a series of studies from our laboratory demonstrating the promise of mouse models to link health effects to biological mechanisms. Specifically, data from 6- to 10-wk-old male ICR mice exposed to intratracheal instillation of 100 μ g of UFPM collected from the Chapel Hill, NC airshed are described. Studies of ischemia/reperfusion, vascular function, and hemostasis are described. In summary, UFPM exposure doubles the size of myocardial infarction attendant to an episode of ischemia and reperfusion while increasing postischemic oxidant stress. UFPM alters endothelial-dependent and -independent regulation of systemic vascular tone; increases platelet number, plasma fibrinogen, and soluble P-selectin levels; and reduces bleeding time, implying enhanced thrombogenic potential. Taking these findings together, this model of acute UFPM exposure in the mouse indicates that UFPM induces a prothrombotic state and decreases vasomotor responsiveness, thereby offering insight into how UFPM could contribute to vascular events associated with thrombosis and ischemia and increasing the extent of infarction.

Chitin particles induce size-dependent but carbohydrate-independent innate eosinophilia.

Murine Mφ that phagocytose CMP develop into M1; this response depends on the size and the chemical composition of the particles. In contrast, recent studies concluded that chitin particles induce M2 and eosinophil migration, promoting acquired Th2 immune responses against chitin‐containing microbes or allergens. This study examined whether these apparently inconsistent responses to chitin could be induced by variation in the size and chemical composition of the chitin particles. We compared the responses of Mφ with CMP, LCB, and Sephadex G‐100 beads (>40 μm). Beads were given i.p. to WT mice and to mice deficient in a CRTH2, a receptor for the eosinophil chemoattractant PGD2. In contrast to the M1 activation induced by CMP, i.p. administration of LCB or Sephadex beads induced within 24 h a CRTH2‐dependent peritoneal eosinophilia, as well as CRTH2‐independent activation of peritoneal Mφ that expressed Arg I, an M2 phenotype. LCB‐induced Mφ exhibited elevated Arg I and a surface MR, reduced surface TLR2 levels, and no change in the levels of CHI3L1 or IL‐10 production. Our results indicate that the effects of chitin in vivo are highly dependent on particle size and that large, nonphagocytosable beads, independent of their chemical composition, induce innate eosinophilia and activate Mφ expressing several M2, but not M1, phenotypes.

PAR-4 Agonist AYPGKF Stimulates Thromboxane Production by Human Platelets

Previous reports have indicated that thrombin-induced thromboxane production by human platelets occurs through two types of interaction between thrombin and the platelet surface. One of these interactions is with protease activated receptor(PAR)-1, the first identified thrombin receptor. These studies were undertaken to determine whether stimulation of PAR-4 also results in thromboxane production. The results show that treatment of washed human platelets with the PAR-4 agonist AYPGKF stimulates a maximum of 40% to 60% of the thromboxane produced by 100 nmol/L thrombin. Maximal thromboxane production requires approximately 1.0 mmol/L AYPGKF, despite the observation that maximal aggregation is produced by 45 &mgr;mol/L AYPGKF. Thromboxane produced by the combined stimulation of PAR-1 and PAR-4 is additive. Pretreatment of platelets with 45 &mgr;mol/L AYPGKF partially desensitizes thromboxane production in response to higher concentrations of AYPGKF and thrombin but not to stimulation by SFLLRN. PAR-4–induced stimulation is also significantly inhibited by 60 &mgr;mol/L genistein. It is concluded that activation through either PAR-1 or PAR-4 results in thromboxane production, but that stimulation of neither receptor alone produces thromboxane equivalent to that produced by 100 nmol/L thrombin. Thus, these findings demonstrate the presence of two pathways for thrombin-induced thromboxane production by platelets as proposed previously.

Thrombin-Induced Thromboxane Synthesis by Human Platelets Properties of an Anion Binding Exosite I– Independent Receptor

These studies have examined the effects of thrombin-related agonists in stimulating thromboxane production by human platelets. The results presented show that (1) the maximal response elicited by thrombin receptor agonist peptide (TRAP) stimulation was 40% to 50% of that seen with thrombin or the thrombin mutant Thrombin Quick I; (2) pretreatment of platelets with prostaglandin E1 or genistein resulted in differential inhibition of thromboxane production in response to TRAP compared with either enzyme agonist; (3) an antibody to the thrombin receptor cleavage site that inhibits increases in intracellular [Ca2+] only partially reduced thromboxane production in response to 5 nmol+L thrombin and 15 nmol/L Thrombin Quick I; (4) preincubation with 20 mumol/L TRAP resulted in desensitization to further stimulation by 100 mumol/L TRAP, but not by 100 nmol/L thrombin; and (5) the response to thrombin after TRAP desensitization was completely inhibited by the tyrosine kinase inhibitor genistein and was independent of an intracellular [Ca2+] flux, The cumulative results may be explained by the existence of two proteolytically activated receptors that result in thromboxane production in response to thrombin. One is the thrombin receptor/substrate, PAR-1. Stimulation through the second receptor/substrate depends on a genistein-sensitive step, is independent of an intracellular Ca2+ flux, and is initiated by a thrombin-activated receptor that does not depend on interaction with anion-binding exosite I, as previously indicated by the relative activity of Thrombin Quick I in stimulating platelet aggregation and thromboxane production. The proposed second thrombin receptor on platelets represents an additional member of the class of proteolytically activated receptors.

Heat-killed BCG induces biphasic cyclooxygenase 2+ splenic macrophage formation—role of IL-10 and bone marrow precursors

Previous studies have shown that prostaglandin E2 (PGE2) release by splenic F4/80+ cyclooxygenase (COX)‐2+ macrophages (MØ) isolated from mice, treated with mycobacterial components, plays a major role in the regulation of immune responses. However, splenic MØ, isolated from untreated mice and treated in vitro with lipopolysaccharide and interferon‐γ, express COX‐1 and COX‐2 within 1 day but release only minimal amounts of PGE2 following elicitation with calcium ionophore A23187. For further characterization of in vivo requirements for development of PGE2‐releasing MØ (PGE2‐MØ), C57Bl/6 [wild‐type (WT)], and interleukin (IL)‐10‐deficient (IL‐10−/−) mice were treated intraperitoneally with heat‐killed Mycobacterium bovis bacillus Calmette‐Guerin (HK‐BCG). One day following injection, COX‐2 was induced in splenic MØ of both mouse strains. However, PGE2 biosynthesis by these MØ was not increased. Thus, expression of COX‐2 is not sufficient to induce PGE2 production in vivo or in vitro. In sharp contrast, 14 days after HK‐BCG treatment, PGE2 release by COX‐2+ splenic MØ increased as much as sevenfold, and a greater increase was seen in IL‐10−/− cells than in WT cells. To further determine whether the 14‐day splenic PGE2‐MØ could be derived from bone marrow precursors, we established a chimera in which bone marrow cells were transfused from green fluorescent protein (GFP)‐transgenic donors to WT mice. Donors and recipients were treated with HK‐BCG simultaneously, and marrow transfusion was performed on Days 1 and 2. On Day 14 after BCG treatment, a significant number of spleen cells coexpressed COX‐2 and GFP, indicating that bone marrow‐derived COX‐2+ MØ may be responsible for the increased PGE2 production.

The many faces of systemic mastocytosis

OBJECTIVE This short review article will augment the readers knowledge of mast cell physiology and will offer an overview of current information on the pathophysiology, heterogeneity, and treatment of human mastocystosis. DATA SOURCES AND STUDY SELECTION Articles published since 1980, textbooks, information from computerized databases, references identified from bibliographies of relevant articles, and books published in the last 10 years. RESULTS AND CONCLUSIONS Mastocytosis is a complex disease with a multitude of clinical presentations, often misdiagnosed, which can embrace characteristics of other diseases and generate a chameleon-like picture. Mast cells possess many important physiologic functions in the human body, but as a consequence of poorly understood events, they can also start a cascade of pathologic reactions. Although a great deal is known about mechanisms involved in physiologic and pathologic processes of mast cells, many areas are waiting to be explored in this millennium.

Effects of protein kinase C inhibitors on thromboxane production by thrombin-stimulated platelets

The purpose of these studies was to identify a possible role for protein kinase C in thromboxane production. The effects of four putative protein kinase C inhibitors were studied with platelet stimulation by thrombin (0.5-150 nM), Thrombin Quick I (1.5-500 nM) or a thrombin receptor (protease activated receptor-1) agonist peptide (TRAP) (5-120 microM). Thromboxane production was increased by the bisindolylmaleimide derivative, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimi de (GF 109203X), unchanged by the inhibitors 12-(2-cyanoethyl)-6,7, 12,13-tetrahydro-13-methyl-5-oxo-5H-indolo (2,3-a) pyrrolo (3, 4-c)-carbazole (Gö 6976) and 5,21:12,17-dimetheno-18H-dibenzo[i, o]pyrrolo[3,4-l][1,8]diazacyclohexadecine-18,20(19H)-dione, 8-[(dimethylamino)methyl]-6,7,8,9,10,11-hexahydro-, monomethanesulfonate (379196), the latter of which is protein kinase C beta-selective, and decreased by 1-[6-[(3-acetyl-2,4, 6-trihydroxy-5-methylphenyl)methyl]-5,7-dihydroxy-2, 2-dimethyl-2H-1-benzopyran-8-yl]-3-phenyl-2-propen-1-one (rottlerin), an inhibitor selective for protein kinase C delta. These results indicate complex regulation of thromboxane synthesis in human platelets including a probable role for protein kinase C delta. The results taken together further suggest that GF 109203X may suppress negative feedback resulting from an unidentified kinase and that the classical protein kinase C isoforms alpha and beta do not have a significant role in regulating thromboxane production by platelets.

Catalytically Inactive Cyclooxygenase 2 and Absence of Prostaglandin E2 Biosynthesis in Murine Peritoneal Macrophages following In Vivo Phagocytosis of Heat-Killed Mycobacterium bovis Bacillus Calmette-Guérin

Over 25 years ago, it was observed that peritoneal macrophages (Mφ) isolated from mice given heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) i.p. did not release PGE2. However, when peritoneal Mφ from untreated mice are treated with HK-BCG in vitro, cyclooxygenase 2 (COX-2), a rate-limiting enzyme for PGE2 biosynthesis, is expressed and the release of PGE2 is increased. The present study of peritoneal Mφ obtained from C57BL/6 mice and treated either in vitro or in vivo with HK-BCG was undertaken to further characterize the cellular responses that result in suppression of PGE2 release. The results indicate that Mφ treated with HK-BCG in vivo express constitutive COX-1 and inducible COX-2 that are catalytically inactive, are localized subcellularly in the cytoplasm, and are not associated with the nuclear envelope (NE). In contrast, Mφ treated in vitro express catalytically active COX-1 and COX-2 that are localized in the NE and diffusely in the cytoplasm. Thus, for local Mφ activated in vivo by HK-BCG, the results indicate that COX-1 and COX-2 dissociated from the NE are catalytically inactive, which accounts for the lack of PGE2 production by local Mφ activated in vivo with HK-BCG. Our studies further indicate that the formation of catalytically inactive COX-2 is associated with in vivo phagocytosis of HK-BCG, and is not dependent on extracellular mediators produced by in vivo HK-BCG treatment. This attenuation of PGE2 production may enhance Mφ-mediated innate and Th1-acquired immune responses against intracellular infections which are suppressed by PGE2.

Differential effects of IL‐10 on prostaglandin H synthase‐2 expression and prostaglandin E2 biosynthesis between spleen and bone marrow macrophages

Different populations of mononuclear phagocytes (MØ) show considerable diversity of cellular function including prostaglandin E2 (PGE2) biosynthesis. Certain bacterial components enhance PGE2 biosynthesis differentially in selected populations of MØ. Interleukin (IL)‐10 is proposed to inhibit modulation of PGE2 biosynthesis by down‐regulating prostaglandin G/H synthase‐2 (PGHS‐2) expression. To assess whether IL‐10 regulates PGE2 biosynthesis and PGHS‐2 expression, splenic and bone marrow MØ were isolated from IL‐10‐deficient (IL‐10−/−), C57Bl/6 [wild‐type (WT) control], and Balb/c (comparison control) mice and were treated with lipopolysaccharide (LPS) and/or interferon‐γ (IFN‐γ) as a model of bacterial inflammation. LPS‐induced PGHS‐2 expression was similar for splenic MØ isolated from the three strains of mice. However, PGE2 released by LPS‐treated splenic MØ was significantly higher in IL‐10−/− and Balb/c than in WT cells. In the presence of LPS and IFN‐γ, PGHS‐2 expression and PGE2 release by IL‐10−/− and Balb/c splenic MØ were enhanced compared with stimulation with LPS alone or IFN‐γ alone. However, there was no significant increase in PGE2 release from WT splenic MØ treated with LPS plus IFN‐γ despite increased PGHS‐2 expression. In sharp contrast, PGHS‐2 expression and PGE2 release by bone marrow MØ were greatly enhanced in IL‐10−/− cells compared with control cells. Our results indicate that IL‐10 regulation of MØ PGE2 biosynthesis and PGHS‐2 expression is compartment‐dependent and that PGE2 production is not linked directly to PGHS‐2 levels. Furthermore, our findings emphasize strain‐specific differences between C57Bl/6 and Balb/c mice, and Balb/c appears more similar to the IL‐10−/− than to the C57Bl/6 with respect to prostanoid production.