Search

Bone marrow (BM) Ang II (angiotensin II) is a major participant in the regulation of hematopoiesis and immunity. The novel tissue substrate Ang-(1–12) [angiotensin-(1–12)] and its cleaving enzyme chymase are an essential source of Ang II production in cardiac tissue. We hypothesized this noncanonical chymase-mediated Ang II–producing mechanism exists in the BM tissue. Immunohistostaining and flow cytometry confirmed the presence of Ang-(1–12) immunoreaction in the BM of SD (Sprague Dawley) rats. Chymase-mediated Ang II–producing activity in BM was ≈1000-fold higher than ACE (angiotensin-converting enzyme)-mediated Ang II–producing activity (4531±137 and 4.2±0.3 fmol/min per mg, respectively; n=6; P P 125 I-Ang II production. Flow cytometry demonstrated that delta median fluorescence intensity of chymase in cluster of differentiation 68 positive cells was significantly higher than that in cluster of differentiation 68 negative cells (1546±157 and 222±48 arbitrary units, respectively; P =0.0021). Cluster of differentiation 68 positive and side scatter low subsets, considered to be myeloid progenitors, express the highest chymase fluorescence intensity in rat BM. Chymase activity and cellular expression was similar in both male and female rats. In conclusion, myeloid lineage cells, especially myeloid progenitors, have an extraordinary Ang II–producing activity by chymase in the BM.

Background: Rat bone marrow (BM)-chimeric experiments reveal a critical role of locally formed angiotensin II (Ang II) in mediating the oxidant-rich inflammatory milieu associated with cardiovascular disease pathogenesis. Extending our first demonstration of renin angiotensin system (RAS) components in the BM, we recently reported a primary role of chymase rather than angiotensin converting enzyme (ACE) in BM generation of Ang II from the novel substrate angiotensin-(1-12) [Ang-(1-12)]. To verify chymase contribution over other proteases such as cathepsins, Ang-(1-12) metabolism studies in rat BM were carried out in the absence and in the presence of a selective orally active chymase inhibitor (TEI-F00806). Methods: Plasma membrane (PM) of BM and left ventricle (LV) tissues were obtained from 12 week-old Sprague Dawley rats following systemic perfusion with phosphate buffered saline with or without the secondary addition of Zamboni’s fixative. PMs were incubated with 125 I-Ang-(1-12) and the metabolic products during incubation were determined by high-performance liquid chromatography. All data were analyzed using GraphPad PRISM 7.0 with P Results: Assessment of 125 I-Ang-(1-12) hydrolysis in the absence of the chymase inhibitor revealed that chymase activity in BM membranes was 280 fold higher than that in LV (4,531 ± 137 fmol/mg/min and 16.3 ± 0.3 fmol/mg/min, respectively, n = 6, P < 0.001). In the presence of TEI-F00806, 96% of the chymase-mediated Ang II production was inhibited in BM. Confocal microscopy showed chymase-positive but tryptase-negative cells as the most dominant in BM, indicating that stem/progenitor cells rather than mature mast cells might be the source of chymase in BM. Conclusion: The current findings demonstrate that BM has tremendously higher chymase-mediated Ang II producing activity from Ang-(1-12) compared to heart tissue. The fact that TEI-00806 inhibited the Ang II producing activity of chymase underscores a tissue-selective enzymatic mechanism for Ang II formation from Ang-(1-12) contributing to the chronic inflammatory processes accompanying the pathogenesis of cardiovascular disease.

Background Activation of the intracrine renin angiotensin systems (RAS) is increasingly recognized as contributing to human pathologies, yet non-canonical renin-independent mechanisms for angiotensin II (Ang II) biosynthesis remain controversial. Direct Ang II generation from angiotensin-(1–12) [Ang-(1–12)] by chymase is an essential intracrine source for regulation of cardiac function. Using a transgenic rat model that overexpresses the human angiotensinogen gene [TGR(hAGT)L1623] and displays increased cardiac Ang II levels, this study aimed to provide evidence for intracrine activation of L-type calcium currents (ICa-L) mediated by the Ang-(1–12)/chymase axis. Methods and results On patch clamp, ICa-L density was significantly higher in TGR(hAGT)L1623 (−6.4 ± 0.3 pA/pF) compared to Sprague Dawley (SD) cardiomyocytes (−4.8, ± 0.5 pA/pF). Intracellular administration of Ang II and Ang-(1–12) elicited a ICa-L increase in both SD and TGR(hAGT)L1623 cardiomyocytes, albeit blunted in transgenic cells. ICa-L activation by intracellular Ang II and Ang-(1–12) was abolished by the specific Ang II type 1 receptor blocker E−3174. Co-administration of a chymase inhibitor prevented activation of ICa-L by Ang-(1–12). Confocal micrographs revealed abundant chymase (mast cell protease 5) immunoreactive protein in SD and TGR(hAGT)L1623 cardiomyocytes. Conclusions Our data demonstrate the existence in cardiomyocytes of a calcium channel modulatory activity responsive to Ang II generated by the Ang-(1–12)/chymase axis that signals via intracellular receptors. Chronically elevated Ang II in TGR(hAGT)L1623 hearts leading to increased intracellular calcium through ICa-L suggests that activation of this Ang-(1–12)/chymase-governed cardiac intracrine RAS may contribute to the pathological phenotypes observed in the humanized model of chronic hypertension and cardiac hypertrophy.

Fully functional CD8+ T cell memory is highly dependent upon CD4+ T cell support. CD4+ T cells play a critical role in inducing the expression of CD70, the ligand for CD27, on dendritic cells. In this study, we demonstrate that CD27 stimulation during primary CD8+ T cell responses regulates the ability to mount secondary CD8+ T cell responses. CD27 stimulation during vaccinia and dendritic cell immunization controls the expression of the IL-7R (CD127), which has been shown to be necessary for memory CD8+ T cell survival. Furthermore, CD27 stimulation during primary CD8+ T cell responses to vaccinia virus restrained the late expression on memory precursor cells of cytokine receptors that support terminal differentiation. The formation of CD8+ T cell memory precursors and secondary CD8+ T cell responses was restored in the absence of CD27 costimulation when endogenous IL-12 was not available. Similarly, the lesion in CD8+ T cell memory that occurs in the absence of CD4+ T cells did not occur in mice lacking IL-12. These data indicate that CD4+ T cell help and, by extension, CD27 stimulation support CD8+ T cell memory by modulating the expression of cytokine receptors that influence the differentiation and survival of memory CD8+ T cells.

The immune response to the tumor can be enhanced by targeting costimulatory molecules on T cells. As the CD70-CD27 costimulatory axis plays an important role in the activation, survival, and differentiation of lymphocytes, we have examined the efficacy of agonistic anti-CD27 antibodies as monotherapies for established melanoma in a murine model. We show that this approach leads to a substantial reduction in the outgrowth of both experimental lung metastases and subcutaneous tumors. Anti-CD27 treatment supports the maintenance of tumor-specific CD8(+) T cells within the tumor, reduces the frequency of FoxP3-expressing CD4(+) T cells within tumors, and potentiates the ability of NK1.1(+) and CD8(+) tumor infiltrating cells to secrete IFNγ upon coculture with tumor cells. The enhanced effector function correlated with lower levels of PD-1 expression on CD8(+) T cells from anti-CD27-treated mice. Despite the modulating effect of anti-CD27 on multiple cell types, only CD8(+) T cells were absolutely required for tumor control. The CD4(+) T cells were dispensable, whereas NK1.1(+) cells were needed during early stages of tumor growth but not for the effectiveness of anti-CD27. Thus, CD27-mediated costimulation provides a potent boost to multiple aspects of the endogenous responses to tumor, and may be exploited to enhance tumor immunity.

Recent data have demonstrated the potential of sphingosine 1-phosphate (S1P) receptor (S1PR) agonism in the treatment of infectious diseases. A previous study used a murine model of Bordetella pertussis infection to demonstrate that treatment with the S1PR agonist AAL-R reduces pulmonary inflammation during infection. In the current study, we showed that this effect is mediated via the S1PR1 on LysM+ (myeloid) cells. Signaling via this receptor results in reduced lung inflammation and cellular recruitment as well as reduced morbidity and mortality in a neonatal mouse model of disease. Despite the fact that S1PRs are pertussis toxin-sensitive G protein-coupled receptors, the effects of AAL-R were pertussis toxin insensitive in our model. Furthermore, our data demonstrate that S1PR agonist administration may be effective at therapeutic time points. These results indicate a role for S1P signaling in B. pertussis-mediated pathology and highlight the possibility of host-targeted therapy for pertussis.

Background— Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) exhibit marked differences in neointimal formation after arterial injury when deficient in apolipoprotein E (apoE −/− ) and fed a Western diet. Quantitative trait locus analysis was performed on an intercross between B6.apoE −/− and C3H.apoE −/− mice to determine genetic factors contributing to the phenotype. Methods and Results— Female B6.apoE −/− mice were crossed with male C3H.apoE −/− mice to generate F 1 s, which were intercrossed to generate 204 male F 2 progeny. At 10 weeks of age, F 2 s underwent endothelium denudation injury to the left common carotid artery. Mice were fed a Western diet for 1 week before and 4 weeks after injury and analyzed for neointimal lesion size, plasma lipid, and membrane cofactor protein (MCP)-1 levels. One significant quantitative trait locus, named Nih1 (61 cM; LOD score, 5.02), on chromosome 12 and a suggestive locus on chromosome 13 (35 cM; LOD score, 2.67) were identified to influence lesion size. One significant quantitative trait locus on distal chromosome 1 accounted for major variations in plasma non–high-density lipoprotein cholesterol and triglyceride levels. Four suggestive quantitative trait locis on chromosomes 1, 2, and 3 were detected for circulating MCP-1 levels. No correlations were observed between neointimal lesion size and plasma lipid levels or between lesion size and plasma MCP-1 levels. Conclusions— Neointimal formation is controlled by genetic factors independent of those affecting plasma lipid levels and circulating MCP-1 levels in the B6 and C3H mouse model.