Aryan M. Namboodiri

Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages.

This study explores the role of mevalonate inhibitors in the activation of NF-kbeta and the induction of inducible nitric oxide synthase (iNOS) and cytokines (TNF-alpha, IL-1beta, and IL-6) in rat primary astrocytes, microglia, and macrophages. Lovastatin and sodium phenylacetate (NaPA) were found to inhibit LPS- and cytokine-mediated production of NO and expression of iNOS in rat primary astrocytes; this inhibition was not due to depletion of end products of mevalonate pathway (e.g., cholesterol and ubiquinone). Reversal of the inhibitory effect of lovastatin on LPS-induced iNOS expression by mevalonate and farnesyl pyrophosphate and reversal of the inhibitory effect of NaPA on LPS-induced iNOS expression by farnesyl pyrophosphate, however, suggests a role of farnesylation in the LPS-mediated induction of iNOS. The inhibition of LPS-mediated induction of iNOS by FPT inhibitor II, an inhibitor of Ras farnesyl protein transferase, suggests that farnesylation of p21(ras) or other proteins regulates the induction of iNOS. Inhibition of LPS-mediated activation of NF-kbeta by lovastatin, NaPA, and FPT inhibitor II in astrocytes indicates that the observed inhibition of iNOS expression is mediated via inhibition of NF-kbeta activation. In addition to iNOS, lovastatin and NaPA also inhibited LPS-induced expression of TNF-alpha, IL-1beta, and IL-6 in rat primary astrocytes, microglia, and macrophages. This study delineates a novel role of the mevalonate pathway in controlling the expression of iNOS and different cytokines in rat astrocytes, microglia, and macrophages that may be important in developing therapeutics against cytokine- and NO-mediated neurodegenerative diseases.

N-ACETYL CYSTEINE INHIBITS INDUCTION OF NO PRODUCTION BY ENDOTOXIN OR CYTOKINE STIMULATED RAT PERITONEAL MACROPHAGES, C6 GLIAL CELLS AND ASTROCYTES

The present study underscores the importance of N-acetyl cysteine (NAC), a potent antioxidant, in inhibiting the induction of NO production by lipopolysaccharides (LPS) and cytokines in peritoneal macrophages, C6 glial cells and primary astrocytes. LPS, interleukin-1 beta (IL-1beta), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) alone or in combinations induced the production of NO to different degrees. NAC when added 2 h earlier to the addition of these stimuli potentially blocked the increase in NO production in macrophages, astrocytes and C6 glial cells. The decrease in NO production by NAC was accompanied by a decrease in inducible nitric oxide synthase (iNOS) activity, in iNOS protein detected by immunoblot analysis with antibodies against iNOS, and in iNOS mRNA determined by reverse-transcriptase coupled polymerase chain reaction (RT-PCR). Time course studies show that inhibition was maximum when NAC was added 2 h prior to the addition of LPS and the degree of inhibition decreased progressively with the increase in time interval when NAC was added after the addition of LPS. In addition to NAC, another antioxidant pyrrolidine dithiocarbamate (PDTC) was also found to inhibit the induction of NO production effectively. Since activation of NF-kappaB is necessary for the induction of iNOS, we examined the effect of NAC on the activation of NF-kappaB. Inhibition of LPS-induced activation of NF-kappaB by NAC in rat peritoneal macrophages suggests that the inhibitory effect of NAC on the induction of iNOS is due to the inhibition of NF-kappaB. Besides NO, NAC also blocked the production of TNF-alpha in rat peritoneal macrophages activated with endotoxin. These results suggest that expression of iNOS and TNF-alpha in macrophages do involve oxygen radicals. The importance of these results in relation to controlling various harmful effects of cytokines released by activated macrophages and glial cells is discussed.

Increasing cAMP Attenuates Induction of Inducible Nitric-oxide Synthase in Rat Primary Astrocytes

Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different brain cells in response to various cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. This study underlines the importance of cAMP in inhibiting the induction of NO production by lipopolysaccharide (LPS) and cytokines in rat primary astrocytes. Compounds (forskolin, 8-bromo-cAMP, and (Sp)-cAMP) that increase cAMP and activate protein kinase A (PKA) were found to inhibit LPS- and cytokine-mediated production of NO as well as the expression of iNOS, whereas compounds (H-89 and (Rp)-cAMP) that decrease cAMP and PKA activity stimulated the production of NO and the expression of iNOS in rat primary astrocytes. Forskolin, but not the inactive analogue 1,9-dideoxyforskolin, inhibited NO production and iNOS expression in a dose-dependent manner in astrocytes. The inhibition of LPS- and/or cytokine-induced NO production in rat C6 glial cells by forskolin suggest that similar to astrocytes, iNOS expression in C6 cells is also regulated by similar mechanisms. In contrast, in rat peritoneal macrophages the cAMP analogues stimulated the LPS- and cytokine-induced production of NO. In vitro, the PKA had no effect on iNOS activity in LPS-treated astrocytes or macrophages, suggesting that PKA modulates the intracellular signaling events associated with the induction of iNOS biogenesis rather than the post-translational modification of iNOS. The compounds which activate PKA activity, blocked the activation of NF-κβ in astrocytes but stimulated the activation of NF-κβ in macrophages. This differential regulation of NF-κβ activation in two different cell types (astrocytes and macrophages) by the same second messenger (cAMP) indicates that intracellular events or pathways in the activation of NF-κβ may be different. Moreover, this inhibition of iNOS expression in LPS- and cytokine-treated astrocytes by cAMP may be of therapeutic potential in NO-mediated cytotoxicity in neurodegenerative diseases.

INHIBITORS OF PROTEIN PHOSPHATASE 1 AND 2A DIFFERENTIALLY REGULATE THE EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN RAT ASTROCYTES AND MACROPHAGES

Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. The present study underlines the importance of protein phosphatase (PP) 1 and 2A in the regulation of the differential expression of iNOS in rat primary astrocytes and macrophages. Compounds (calyculin A, microcystin, okadaic acid, and cantharidin) that inhibit PP 1 and 2A were found to stimulate the lipopolysaccharide (LPS)- and cytokine-mediated expression of iNOS and production of NO in rat primary astrocytes and C6 glial cells. However, these inhibitors inhibited the LPS- and cytokine-mediated expression of iNOS and production of NO in rat resident macrophages and RAW 264.7 cells. Similarly, okadaic acid, an inhibitor of PP 1/2A, stimulated the iNOS promoter-derived chloramphenicol acetyltransferase activity in astrocytes and inhibited the iNOS promoter-derived chloramphenicol acetyltransferase activity in macrophages, indicating that okadaic acid also differentially regulates the transcription of the iNOS gene in astrocytes and macrophages. The observed stimulation of the expression of iNOS in astrocytes and the inhibition of the expression of iNOS in macrophages with the inhibition of PP 1/2A activity clearly delineate a novel role of PP 1/2A in the differential regulation of iNOS in rat astrocytes and macrophages. Because the activation of NF-κB is necessary for the induction of iNOS and the expression of tumor necrosis factor (TNF)-α also depends on the activation of NF-κB, we examined the effect of okadaic acid on the LPS-mediated activation of NF-κB and production of TNF-α in rat primary astrocytes and macrophages. Interestingly, in both cell types, okadaic acid stimulated the LPS-mediated DNA binding as well as transcriptional activity of NF-κB and production of TNF-α. This study suggests that the stimulation of iNOS expression in astrocytes by inhibitors of PP 1/2A is possibly due to the stimulation of NF-κB activation; however, activation of NF-κB is not sufficient for the induction of iNOS in macrophages and that apart from NF-κB some other signaling pathway(s) sensitive to PP 1 and/or PP 2A is/are possibly involved in the regulation of iNOS in macrophages. This differential induction of iNOS as compared with similar activation of NF-κB by inhibitors of PP 1/2A indicates the involvement of different intracellular signaling events for the induction of iNOS in two cell types of the same animal species.

IgG antibodies to human cytomegalovirus late protein UL94 in patients with systemic sclerosis

Human cytomegalovirus (HCMV) has been proposed as an amplifying agent for at least some of the spectrum of systemic sclerosis (SSc; scleroderma). In support of this hypothesis, antibodies to the HCMV late protein UL94 have been detected in the majority of SSc patients in a study involving Caucasian subjects from Italy. The aim of this investigation was to determine whether elevated levels of anti-UL94 antibodies are present in African American and Caucasian SSc patients from the U.S. We further wished to determine whether there was a significant difference in the levels of anti-UL94 antibodies between the diffuse and the limited forms of the disease. IgG antibodies to a UL94 peptide were measured in 254 Caucasian and 90 African American subjects by an enzyme-linked immunosorbent assay (ELISA). In both Caucasian and African American subjects, the mean antibody level in the diffuse form of SSc was significantly higher than that in the respective control subjects (714 vs. 466 ng/ml, p=0.005; 1226 vs. 512 ng/ml, p<0.0001). Also, among Caucasian SSc patients, the mean antibody level in the diffuse form of SSc was significantly higher than that in the limited form of the disease (714 vs. 465 ng/ml, p=0.02). These results show that increased levels of antibodies to the HCMV late protein UL94 are associated with SSc and they may be a marker for the severity of the disease.

Genetic Markers of IgG Influence the Outcome of Infection with Hepatitis C Virus

We examined the role that immunoglobulin GM and KM allotypes-genetic markers of gamma and kappa chains, respectively-play in the outcome of hepatitis C virus (HCV) infection in white Americans. A total of 119 persons who had cleared HCV and 111 with persistent HCV infection were genotyped for the presence of several GM and KM determinants. Persistent HCV infection was more than three times as likely (odds ratio, 3.50; P= .01) in subjects who were carriers of the GM3 allele than in those who were noncarriers. These results show that particular GM alleles may be important determinants of the outcome of HCV infection.

IGKC and FcγR genotypes and humoral immunity to HER2 in breast cancer.

Immunoglobulin κ constant (IGKC) gene has recently been identified as a strong prognostic marker in several human solid tumors, including breast cancer. Although the mechanisms underlying the IGKC signature are not yet known, identification of tumor-infiltrating plasma cells as the source of IGKC expression strongly suggests a role for humoral immunity in breast cancer progression. The primary aim of the present investigation was to determine whether the genetic variants of IGKC, KM (κ marker) allotypes, are risk factors for breast cancer, and whether they influence the magnitude of humoral immunity to epidermal growth factor receptor 2 (HER2), which is overexpressed in 25-30% of breast cancer patients and is associated with poor prognosis. Using a matched case-control design, we genotyped a large (1719 subjects) study population from Japan and Brazil for KM alleles. Both cases and controls in this study population had been previously characterized for GM (γ marker) and Fcγ receptor (FcγR) alleles, and the cases had also been characterized for anti-HER2 antibodies. Conditional logistic regression analysis of the data showed that KM1 allele additively contributed to the risk of breast cancer in the Japanese subjects from Nagano: Compared to KM3 homozygotes, KM1 homozygotes were almost twice as likely to develop breast cancer (OR=1.77, CI 1.06-2.95). Additionally, KM genotypes-individually and in particular epistatic combinations with FcγRIIa genotypes-contributed to the magnitude of anti-HER2 antibody responsiveness in the Japanese patients. This is the first report implicating KM alleles in the immunobiology of breast cancer.

Immunoglobulin genes and the acquisition of HIV infection in a randomized trial of recombinant adenovirus HIV vaccine.

Our knowledge of the host genetic factors that contribute to the acquisition of HIV infection is limited. To identify the host genetic correlates of HIV1 acquisition, we genotyped 777 participants of a randomized trial of recombinant adenovirus HIV1 vaccine for Fcγ receptor IIa (FcγRIIa), FcγRIIIa, and several GM and KM alleles-genetic markers of immunoglobulin γ and κ chains, respectively. None of the genotypes by itself was significantly associated with the acquisition of HIV1 infection. However, particular combinations of GM and KM as well as those of GM and FcγRIIIa loci were significantly associated with the acquisition of HIV1 infection epistatically: KM1/3-GM3/17 (interaction p=0.0246; FDR=0.2952), KM1/3-GM5/21 (interaction p=0.0016; FDR=0.0960), and GM23+/-FcγRIIIa (interaction p=0.0060; FDR=0.1200). These results suggest the involvement of GM, KM, and FcγRIIIa loci in the acquisition of HIV infection. Additional studies are warranted.

Racially restricted contribution of immunoglobulin Fcγ and Fcγ receptor genotypes to humoral immunity to human epidermal growth factor receptor 2 in breast cancer

Tumour‐associated antigen human epidermal growth factor receptor 2 (HER2) is over‐expressed in 25–30% of breast cancer patients and is associated with poor prognosis. Naturally occurring anti‐HER2 antibody responses have been described in patients with HER2 over‐expressing tumours. There is significant interindividual variability in antibody responsiveness, but the host genetic factors responsible for this variability are poorly understood. The aim of the present investigation was to determine whether immunoglobulin genetic markers [GM (genetic determinants of γ chains)] and Fcγ receptor (FcγR) alleles contribute to the magnitude of natural antibody responsiveness to HER2 in patients with breast cancer. A total of 855 breast cancer patients from Japan and Brazil were genotyped for several GM and FcγR alleles. They were also characterized for immunoglobulin (Ig)G antibodies to HER2. In white subjects (n = 263), GM 23‐carriers had higher levels of anti‐HER2 antibodies than non‐carriers of this allele (p = 0·004). At the GM 5/21 locus, the homozygotes for the GM 5 allele had higher levels of anti‐HER2 antibodies than the other two genotypes (P = 0·0067). In black subjects (n = 42), FcγRIIa‐histidine/histidine homozygotes and FcγRIIIa‐phenylalanine/valine heterozygotes were associated with high antibody responses (P = 0·0071 and 0·0275, respectively). FcγR genotypes in white subjects and GM genotypes in black subjects were not associated with anti‐HER2 antibody responses. No significant associations were found in other study groups. These racially restricted contributions of GM and FcγR genotypes to humoral immunity to HER2 have potential implications for immunotherapy of breast cancer.

Transcriptional regulation of the rat eIF4E gene in cardiac muscle cells: the role of specific elements in the promoter region.

Eukaryotic initiation factor 4E (eIF4E) binds to the 7-methylguanosine cap of mRNA and facilitates binding of mRNA to the 40 S ribosome, a rate-limiting step in translation initiation. The expression of eIF4E mRNA and protein increases during growth of cardiac muscle cells (cardiocytes) in vitro. To examine transcriptional regulation of the rat eIF4E gene, 2.1 kB of the rat eIF4E promoter region was cloned and the contribution of specific elements in regulating transcription was determined in primary cultures of rat cardiocytes and in a murine C(2)C(12) myoblast cell line. Sequence analysis of the rat eIF4E promoter revealed 80% sequence similarity with human eIF4E. A putative distal E-box was found at -230 bp and a proximal E-box was located at -77 bp upstream of the transcription start site. Consensus AP-1 motifs were found at -839 and -901 bp and designated as the proximal AP-1 site and distal AP-1 site, respectively. Transfection of reporter gene constructs into cardiocytes showed that deletion of the region between -633 and -318 bp produced a 3-fold increase in basal transcription as compared to the 2.1 kB eIF4E promoter construct. Further deletion of the distal E-box region had no effect on transcription as compared with the 2.1 kB promoter, but deletion of both E-boxes eliminated transcriptional activity. Similar results were obtained in C(2)C(12) myoblasts. To further investigate transcriptional regulation, point mutations were made in the 2.1 kB eIF4E promoter. Mutation of either the distal or proximal E-box had minimal effects on activity in either cell type, but mutation of the distal AP-1 site significantly reduced eIF4E promoter activity by 66+/-4% in cardiocytes. In C(2)C(12) myoblasts, mutating the distal AP-1 site reduced activity by 30+/-4% We conclude that both E-boxes are required for maximal basal activity of the eIF4E promoter, and that the distal AP-1 motif may activate transcription.