John S.D. Chan

Macrophages and control of granulomatous inflammation in tuberculosis

The granuloma that forms in response to Mycobacterium tuberculosis must be carefully balanced in terms of immune responses to provide sufficient immune cell activation to inhibit the growth of the bacilli, yet modulate the inflammation to prevent pathology. There are likely many scenarios by which this balance can be reached, given the complexity of the immune responses induced by M. tuberculosis. In this review, we focus on the key role of the macrophage in balancing inflammation in the granuloma.

The Inducible Nitric Oxide Synthase Locus Confers Protection against Aerogenic Challenge of Both Clinical and Laboratory Strains of Mycobacterium tuberculosis in Mice

ABSTRACT Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) against Mycobacterium tuberculosisinfection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance of RNI in host defense against M. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous,M. tuberculosis infection of mice. Furthermore, in vitro studies have demonstrated that different strains of M. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought to test rigorously the protective role of RNI against infection with recent clinical isolates of M. tuberculosis following both aerogenic and intravenous challenges. Three recently isolated and unique M. tuberculosis strains were used to infect both wild-type (wt) C57BL/6 and NOS2 gene-disrupted mice. Regardless of the route of infection, NOS2−/− mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory strain of M. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2−/− mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2−/− mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required for the control of murine tuberculous infection caused by both laboratory and clinical strains of M. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.

Loss of Pentameric Symmetry of C-reactive Protein Is Associated with Delayed Apoptosis of Human Neutrophils

Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. Here we report that a novel signal for delaying neutrophil apoptosis is the classic acute phase reactant, C-reactive protein (CRP). However, this anti-apoptotic activity is expressed only when the cyclic pentameric structure of CRP is lost, resulting in formation of modified or monomeric CRP (mCRP), which may be formed in inflamed tissues. By contrast, native pentameric CRP and CRP peptides 77–82, 174–185, and 201–206 failed to affect neutrophil apoptosis. The apoptosis delaying action of mCRP was markedly attenuated by an antibody against the low affinity IgG immune complex receptor (CD16) but not by an anti-CD32 antibody. mCRP evoked a transient concurrent activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways, leading to inhibition of caspase-3 and consequently to delaying apoptosis. Consistently, pharmacological inhibition of either ERK or Akt reversed the anti-apoptotic action of mCRP; however, they did not produce additive inhibition. Thus, mCRP, but not pentameric CRP or peptides derived from CRP, promotes neutrophil survival and may therefore contribute to amplification of the inflammatory response.

Attenuation of Interstitial Fibrosis and Tubular Apoptosis in db/db Transgenic Mice Overexpressing Catalase in Renal Proximal Tubular Cells

OBJECTIVE—The present study investigated the relationships between reactive oxygen species (ROS), interstitial fibrosis, and renal proximal tubular cell (RPTC) apoptosis in type 2 diabetic db/db mice and in db/db transgenic (Tg) mice overexpressing rat catalase (rCAT) in their RPTCs (db/db rCAT-Tg). RESEARCH DESIGN AND METHODS—Blood pressure, blood glucose, and albuminuria were monitored for up to 5 months. Kidneys were processed for histology and apoptosis studies (terminal transferase-mediated dUTP nick-end labeling or immunostaining for active caspase-3 and Bax). Real-time quantitative PCR assays were used to quantify angiotensinogen (ANG), p53, and Bax mRNA levels. RESULTS—db/db mice developed obesity, hyperglycemia, hypertension, and albuminuria. In contrast, db/db rCAT-Tg mice became obese and hyperglycemic but had normal blood pressure and attenuated albuminuria compared with db/db mice. Kidneys from db/db mice displayed progressive glomerular hypertrophy, glomerulosclerosis, interstitial fibrosis, and tubular apoptosis and increased expression of collagen type IV, Bax, and active caspase-3, as well as increased ROS production. These changes, except glomerular hypertrophy, were markedly attenuated in kidneys of db/db rCAT-Tg mice. Furthermore, ANG, p53, and Bax mRNA expression was increased in renal proximal tubules of db/db mice but not of db/db rCAT-Tg mice. CONCLUSIONS—Our results indicate a crucial role for intra-renal ROS in the progression of hypertension, albuminuria, interstitial fibrosis, and tubular apoptosis in type 2 diabetes and demonstrate the beneficial effects of suppressing ROS formation.

Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1[1–32]

We recently reported that matrix metal‐loproteinase 2 (MMP‐2, gelatinase A) cleaves big endo‐thelin 1 (ET‐1), yielding the vasoactive peptide ET‐1[1–32]. We tested whether ET‐1[1–32] could affect the adhesion of human neutrophils to coronary artery endothelial cells (HCAEC). ET‐1[1–32] rapidly down‐regulated the expression of L‐selectin and up‐regulated expression of CD11b/CD18 on the neutrophil surface, with EC50 values of 1–3 nM. These actions of ET‐1[1–32] were mediated via ETA receptors and did not require conversion of ET‐1[1–32] into ET‐1 by neutrophil proteases, as revealed by liquid chromatography and mass spectroscopy. Moreover, ET‐1[1–32] evoked release of neutrophil gelatinase B, which cleaved big ET‐1 to yield ET‐1[1–32], thus revealing a positive feedback loop for ET‐1[1–32] generation. Up‐regula‐tion of CD11b/CD18 expression and gelatinase release was tightly associated with activation of extracellular signal‐regulated kinase (Erk). Stimulation of Erk activity was due to activation of Ras, Raf‐1, and MEK (MAPK kinase). ET‐1[1–32] also produced slight increases in the expression of ICAM‐1 and E‐selectin on HCAEC, and markedly enhanced ß2 integrin‐dependent adhesion of neutrophils to activated HCAEC. These results are the first indication that gelatinolytic MMPs via cleavage of big ET‐1 to yield ET‐1[1–32] activate neu‐trophils and promote leukocyte‐endothelial cell adhesion and, consequently, neutrophil trafficking into inflamed tissues.—Fernandez‐Patron, C., Zouki, C., Whittal, R., Chan, J. S. D., Davidge, S. T., Filep, J. G. Matrix metalloproteinases regulate neutrophil‐endothelial cell adhesion through generation of endothelin‐ 1[1–32]. FASEB J. 15, 2230–2240 (2001)

Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo

ABSTRACT Mycobacterium tuberculosis possesses five genes with significant homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. The M. luteus Rpf is a secreted ∼16-kDa protein which restores active growth to cultures of M. luteus rendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins of M. tuberculosis have been shown to stimulate the growth of extended-stationary-phase cultures of Mycobacterium bovis BCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function of M. tuberculosis Rpf homologues in vivo. To address these questions, we have disrupted each of the five rpf-like genes in M. tuberculosis Erdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast to M. luteus, for which rpf is an essential gene, we find that all of the M. tuberculosis rpf deletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis of rpf expression in M. tuberculosis cultures from early log phase through late stationary phase indicates that expression of the rpf-like genes is growth phase-dependent, and that the expression patterns of the five M. tuberculosis rpf genes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterial rpf genes are expressed in vivo in the lungs of mice acutely infected with virulent M. tuberculosis.

Effect of Paricalcitol on Left Ventricular Mass and Function in CKD—The OPERA Trial

Vitamin D seems to protect against cardiovascular disease, but the reported effects of vitamin D on patient outcomes in CKD are controversial. We conducted a prospective, double blind, randomized, placebo-controlled trial to determine whether oral activated vitamin D reduces left ventricular (LV) mass in patients with stages 3-5 CKD with LV hypertrophy. Subjects with echocardiographic criteria of LV hypertrophy were randomly assigned to receive either oral paricalcitol (1 μg) one time daily (n=30) or matching placebo (n=30) for 52 weeks. The primary end point was change in LV mass index over 52 weeks, which was measured by cardiac magnetic resonance imaging. Secondary end points included changes in LV volume, echocardiographic measures of systolic and diastolic function, biochemical parameters of mineral bone disease, and measures of renal function. Change in LV mass index did not differ significantly between groups (median [interquartile range], -2.59 [-6.13 to 0.32] g/m(2) with paricalcitol versus -4.85 [-9.89 to 1.10] g/m(2) with placebo). Changes in LV volume, ejection fraction, tissue Doppler-derived measures of early diastolic and systolic mitral annular velocities, and ratio of early mitral inflow velocity to early diastolic mitral annular velocity did not differ between the groups. However, paricalcitol treatment significantly reduced intact parathyroid hormone (P<0.001) and alkaline phosphatase (P=0.001) levels as well as the number of cardiovascular-related hospitalizations compared with placebo. In conclusion, 52 weeks of treatment with oral paricalcitol (1 μg one time daily) significantly improved secondary hyperparathyroidism but did not alter measures of LV structure and function in patients with severe CKD.

Tissue Distribution and Molecular Forms of a Novel Pituitary Protein in the Rat

A sensitive and specific radioimmunoassay (RIA) was developed for a novel pituitary protein that we recently isolated from human and porcine pituitary gland and designated 7B2. By employing this RIA, we were able to detect and assay this novel protein in different rat tissue extracts. The concentrations of 7B2 in rat anterior pituitary lobe, neurointermediate lobe, hypothalamus, adrenal medulla and thyroid gland were 10,400 +/- 804; 6,190 +/- 908; 773 +/- 50; 697 +/- 83 and 1,368 +/- 116 pg/mg tissue (wet weight, n = 10, mean +/- SEM), respectively. However, the concentrations of 7B2 were lower than 30 pg/mg tissue in extracts of pancreas, ileum and colon, and were below the sensitivity of the RIA in extracts of liver, kidney, spleen, lung, adrenal cortex and testis. Gel permeation chromatography of extracts of anterior pituitary lobe, neurointermediate lobe, hypothalamus, adrenal medulla and thyroid gland on Sephadex G-100 revealed that most of the immunoreactive (Ir)-7B2 has an apparent molecular weight of 45,000-50,000. Subsequent dissociation of this Ir-7B2 by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS) yielded an Ir-7B2 with an apparent molecular weight of around 19,000. In addition, high K+ concentration (50 mM) induced the release of Ir-7B2 from cultured cells of both rat anterior pituitary and neurointermediate lobe. Finally, Ir-7B2 was detected in the neurosecretory granule fraction prepared from porcine neurointermediate lobe. These results indicate that 7B2 may be a novel secretory protein in the pituitary gland.

Peroxynitrite induces integrin-dependent adhesion of human neutrophils to endothelial cells via activation of the Raf-1/MEK/Erk pathway

Accumulating evidence suggests that enhanced peroxynitrite (ONOO‐) formation occurs during inflammation. We have studied the impact and the mechanisms of ONOO‐ action on expression of adhesion molecules on human neutrophils and coronary artery endothelial cells (HCAEC) and binding of neutrophils to HCAEC. Addition of ONOO‐ (0.1 to 200 µΜ) to isolated neutrophils resulted in a concentration‐dependent down‐regulation of L‐selectin expression, and up‐regulation of CD11b/CD18 expression. ONOO‐ stimulation of Erk activity was accompanied by activation of Ras, Raf‐1 and MEK (mitogen‐activated protein kinase kinase), and was sensitive to the MEK inhibitor PD 98059. We have observed a tight association between Erk activation and changes in CD11b/CD18 expression. ONOO‐ also evoked activation of neutrophil p38 MAPK. Neither ONOO‐‐induced up‐regulation of CD11b/CD18 expression nor Erk activation was affected by SB 203580, a selective inhibitor of p38 MAPK. ONOO‐ by itself had little effect on expression of ICAM‐1 and E‐selectin on HCAEC, whereas it markedly enhanced attachment of neutrophils to lipopolysaccharide‐activated HCAEC only when it was added together with neutrophils. Increases in neutrophil adhesion evoked by ONOO‐ were blocked by an anti‐CD18 monoclonal antibody. These data suggest that ONOO‐ activates Erk in neutrophils via the Ras/Raf‐1/MEK signal transduction pathway, leading to up‐regulation of surface expression of CD11b/CD18 and consequently to increased neutrophil adhesion to endothelial cells.

Overexpression of Angiotensinogen Increases Tubular Apoptosis in Diabetes

The intrarenal renin-angiotensin system (RAS) plays an important role in the progression of diabetic nephropathy. We have previously reported that mice overexpressing angiotensinogen in renal proximal tubular cells (RPTC) develop hypertension, albuminuria, and renal injury. Here, we investigated whether activation of the intrarenal RAS contributes to apoptosis of RPTC in diabetes. Induction of diabetes with streptozotocin in these transgenic mice led to significant increases in BP, albuminuria, RPTC apoptosis, and proapoptotic gene expression compared with diabetic nontransgenic littermates. Insulin and/or RAS blockers markedly attenuated these changes. Hydralazine prevented hypertension but not albuminuria, RPTC apoptosis, or proapoptotic gene expression. In vitro, high-glucose medium significantly increased apoptosis and caspase-3 activity in rat immortalized RPTC overexpressing angiotensinogen compared with control cells, and these changes were prevented by insulin and/or RAS blockers. In conclusion, intrarenal RAS activation and high glucose may act in concert to increase tubular apoptosis in diabetes, independent of systemic hypertension.