Sastry Gollapudi

Impaired Functions of Peripheral Blood Monocyte Subpopulations in Aged Humans

Aging is associated with increased susceptibility to microbial infections, and monocytes play an important role in microbial defense. In this study, we have identified and compared four subpopulations of monocytes (CD14++(high)CD16−, CD14+(low)CD16−, CD14++(high)CD16+, and CD14+(low)CD16+) in the peripheral blood of young and aged subjects with regard to their numbers, cytokine production, TLR expression, and phosphorylation of ERK1/2 in response to pam3Cys a TLR-1/2 ligand. Proportions and numbers of CD14++(high)CD16+ and CD14+(low)CD16+ monocytes were significantly increased, whereas proportions of CD14+(low)CD16− monocytes were decreased in aged subjects as compared to young subjects. In aged subjects, IL-6 production by all four subsets of monocytes was significantly decreased, whereas TNF-α production was decreased in monocyte subsets, except the CD14+(low)CD16− subset. A significantly reduced expression of TLR1 was observed in CD14++(high)CD16+ and CD14+(low)CD16+ monocyte subsets in aged subjects. Furthermore, following pam3Cys stimulation, ERK1/2 phosphorylation was significantly lower in CD14+(low)CD16+, CD14++(high)CD16+, and CD14+(low)CD16− subsets of monocytes from aged subjects. This is the first study of four subpopulations of monocytes in aging, which demonstrates that their functions are differentially impaired with regard to the production of cytokines, expression of TLR, and signaling via the ERK–MAPK pathway. Finally, changes in the number of monocyte subsets, and impairment of TLR1 expression, TNF-α production, and EK1/2 phosphorylation was more consistent in CD16+ monocyte subsets regardless of expression of CD14high or CD14+low, therefore highlighting the significance of further subdivision of monocytes into four subpopulations.

Preferential expression and activity of multidrug resistance gene 1 product (P-glycoprotein), a functionally active efflux pump, in human CD8 + T cells: A role in cytotoxic effector function

The multidrug resistance gene 1 (mdr 1) product, the P-glycoprotein (Pgp), is a 170-kD transmembrane transport protein, whose overexpression is associated with multidrug resistance in cancer cells and in chloroquine-resistantPlasmodium falciparum infection. In this study we show that normal freshly isolated human lymphocytes express low levels ofmdr 1 mRNA and membrane Pgp. Although Pgp is expressed in both CD4+ and CD8+ T cells, it is preferentially expressed in CD8+ T cells. Activation of T lymphocytes with phytohemagglutinin leads to an amplification of bothmdr 1 mRNA and membrane Pgp in T cells. P-glycoprotein in T cells is a functionally active efflux pump as demonstrated by decreased retention of rhodamine-123 and its increased accumulation by cyclosporin A, an inhibitor of Pgp function. In addition, MRK-16 antibody increased accumulation of Rh123 in CD8+ T cells. Furthermore, MRK16 anti-P-glycoprotein monoclonal antibody, in a concentration-dependent manner, inhibited T lymphocyte-mediated cytotoxicity. These data suggest a physiologic role of P-glycoprotein in cytotoxic T-lymphocyte effector function.

Leptin Activates Human B Cells to Secrete TNF-α, IL-6, and IL-10 via JAK2/STAT3 and p38MAPK/ERK1/2 Signaling Pathway

Leptin, one of the adipokines, functions as a hormone and a cytokine. In this investigation, we show for the first time that leptin, in a concentration-dependent manner, activates human peripheral blood B cells to induce secretion of IL-6, IL-10, and TNF-α. Leptin increased B cells expressing CD25 and HLA-DR. Leptin induces phosphorylation of Janus activation kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), p38 mitogen-activated protein kinase (p38MAPK), and extracellular signal-regulated kinase (ERK1/2). Furthermore, leptin-induced cytokine secretion by B cells was blocked by inhibitors of JAK2, STAT3, p38MAPK, and ERK1/2. These data demonstrate that leptin activates human B cells to secrete cytokines via activation of JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathways, which may contribute to its inflammatory and immunoregulatory properties.

Characterization of naı̈ve, memory and effector CD8+ T cells: effect of age

Aging is associated with progressive decline in T cell functions and increased frequency of infections, autoimmune phenomenon, and cancer. Memory T cells rapidly acquire effector functions to kill infected and malignant cells and/or inhibit their replication. Recently, memory T cells have been further classified into central and effector memory T cells (and early and intermediate T cells by some investigators). In aging, memory T cells are accumulated; however, these subpopulations of memory and effector T cells have not been fully characterized and changes in central memory and effector memory T cells in aged humans have not been described. In this article, we have further defined naïve, central memory, effector memory, and effector CD8+ T cells in humans and their changes in aged humans.

P-glycoprotein (MDR 1 gene product) in cells of the immune system: its possible physiologic role and alteration in aging and human immunodeficiency virus-1 (HIV-1) infection.

P-glycoprotein, a 170-kd glycoprotein encoded by theMDR 1 gene, is a member of a highly conserved superfamily of ATP-binding cassette (ABC) transport proteins. It shares extensive homology with numerous bacterial and eukaryotic ABC transport proteins. P-glycoprotein acts as an energy-dependent efflux pump that appears to transport structurally diverse agents ranging from ions to peptides. P-glycoprotein (P-gP) has been implicated as playing a role in multidrug (MDR) resistance in cancer, chloroquine-resistantPlasmodium falciparum infection, and possibly human immunodeficiency virus-1 (HIV-1) resistance to nucleoside compounds. A number of normal tissues in humans and rodents have been shown to express high levels of P-gp. The expression and function of P-gp in cells of the immune system have been explored in the past 2 years. This review presents a state of the art regarding the expression, regulation, and function of Pgp in cells of the immune system. In addition, its alteration in aging and HIV-1 infection is reviewed. A possible physiologic role of P-gp in cytokine secretion, antigen processing/presentation, and effector functions is also discussed.

Functional decline in aging and disease: a role for apoptosis.

The process of aging and senescence is associated with a decline in several organ functions and ultimately takes away independence and reduces quality of life. The precariously marginal functional reserves of the immune, pulmonary, and cardiovascular systems are among the most important causes of increased hospitalization in the older population. When complicated by chronic diseases, as is often the case, the problem is magnified. Apoptosis, or programmed cell death, is a process that goes on continuously throughout life. It is involved in embryogenesis for proper organ and tissue development. After birth and through adulthood, it helps eliminate unneeded and damaged cells. There is evidence that advanced age is associated with dysregulation of apoptosis. Several studies have shown age‐related changes in the levels of proteins and factors that regulate apoptosis. This could explain the age‐associated increased prevalence of cancers, certain autoimmune diseases, and neurodegenerative disorders in older people. More studies are needed to further elucidate the process of apoptosis. With this knowledge, the use of gene therapy and apoptosis modulators may someday have therapeutic value in preventing the functional decline we see in the older population.

Effect of end-stage renal disease on B-lymphocyte subpopulations, IL-7, BAFF and BAFF receptor expression.

Background. End-stage renal disease (ESRD) results in increased susceptibility to infections, impaired response to vaccination and diffuse B-cell lymphopenia. However, the precise nature and mechanism of ESRD-induced B-cell lymphopenia remains unclear. Therefore, we studied the distribution of major B-cell subsets, B-cell growth, differentiation and survival factors, IL-7 and BAFF, and their receptors in 21 haemodialysis patients and 21 controls. Methods. Innate B1 cells (CD19+, CD5+), conventional B2 cells (CD19+, CD5−), newly formed transitional B cells (CD19+, CD10+, CD27−), naïve B cells (CD19+, CD27−) and memory B cells (CD19+, CD27+) and BAFF receptor were quantified by flow cytometry. Plasma IL-7, BAFF, IL-6, TNF-α and IL-10 were measured by ELISA. Results. The ESRD group exhibited significant reductions of all B-cell subpopulations except for transitional B cells that were less severely affected. No significant difference was found in B-cell apoptosis between the ESRD and control groups. Moreover, plasma IL-7 and BAFF levels were elevated in ESRD patients, therefore excluding their deficiencies as a possible culprit. However, BAFF receptor expression was significantly reduced in transitional but not mature B cells in the ESRD group. Interestingly, B-cell activation with the TLR9 agonist resulted in significantly greater production of IL-6 and TNF alpha but not IL-10 in the ESRD group. Conclusions. Thus, despite elevation of B-cell growth, differentiation and survival factors, ESRD patients exhibited diffuse reduction of B-cell subpopulations. This was associated with the down-regulation of BAFF receptor in transitional B cells. The latter can, in part, contribute to B-cell lymphopenia by promoting resistance to the biological actions of BAFF that is a potent B-cell differentiation and survival factor.

Modified arabinoxylan rice bran (MGN-3/Biobran) sensitizes human T cell leukemia cells to death receptor (CD95)-induced apoptosis

MGN-3, an arabinoxylan extracted from rice bran that is treated enzymatically with an extract from Shiitaki mushrooms, is an effective biological response modifier that increases NK cell activity, and potentiates the activity of conventional chemotherapeutic agents. In this study, we investigated the effect of MGN-3 on death receptor-induced apoptosis in the human leukemic HUT 78 cell line. HUT 78 cells were pre-treated with MGN-3, and then were incubated with the agonistic antibody against death receptor (Fas, CD95). Apoptosis was determined by the propidium iodide technique using FACScan. Activation of caspase 3, caspase 8, and caspase 9 was determined by flow cytometry. Mitochondrial membrane potential was measured with DIOC(6) dye using FACScan. Expression of CD95 and Bcl-2 were measured by flow cytometry. In a dose-dependent manner, MGN-3 enhanced anti-CD95 antibody-induced apoptosis. Increased cell death was correlated with increased depolarization of mitochondrial membrane potential and increased activation of caspase 3, caspase 8, and caspase 9. MGN-3 treatment had no effect on the level of expression of CD95, but it caused down regulation of Bcl-2 expression. These results suggest that MGN-3 increases the susceptibility of cancer cells to undergo apoptosis mediated by death ligands, which may be relevant for anti-cancer activities.

Thimerosal induces TH2 responses via influencing cytokine secretion by human dendritic cells.

Thimerosal is an organic mercury compound that is used as a preservative in vaccines and pharmaceutical products. Recent studies have shown a TH2‐skewing effect of mercury, although the underlying mechanisms have not been identified. In this study, we investigated whether thimerosal can exercise a TH2‐promoting effect through modulation of functions of dendritic cells (DC). Thimerosal, in a concentration‐dependent manner, inhibited the secretion of LPS‐induced proinflammatory cytokines TNF‐α, IL‐6, and IL‐12p70 from human monocyte‐derived DC. However, the secretion of IL‐10 from DC was not affected. These thimerosal‐exposed DC induced increased TH2 (IL‐5 and IL‐13) and decreased TH1 (IFN‐γ) cytokine secretion from the T cells in the absence of additional thimerosal added to the coculture. Thimerosal exposure of DC led to the depletion of intracellular glutathione (GSH), and addition of exogenous GSH to DC abolished the TH2‐promoting effect of thimerosal‐treated DC, restoring secretion of TNF‐α, IL‐6, and IL‐12p70 by DC and IFN‐γ secretion by T cells. These data suggest that modulation of TH2 responses by mercury and thimerosal, in particular, is through depletion of GSH in DC.

Leukocyte Toll-Like Receptor Expression in End-Stage Kidney Disease

Background: End-stage renal disease (ESRD) is simultaneously associated with inflammation, impaired immunity and increased susceptibility to microbial infections. Innate immune cells, monocytes and polymorphonuclear leukocytes (PMN) recognize pathogens via toll-like receptors (TLR) triggering phagocytosis, cellular activation and secretion of inflammatory cytokines. Data on expression and function of TLRs in ESRD are limited. Methods: Blood samples from 21 stable ESRD patients and 21 normal controls were processed for TLR2, TLR4, TLR7 and TLR 9 expression on monocytes and PMN by flow cytometry. TLR activity was examined by determining the response to TLR4 and TLR2 ligands. Results: The ESRD group exhibited significant upregulation of TLR2 and TLR4 (but not TLR7 or TLR 9) expressions on monocytes and of TLR4 on PMN. This was coupled with heightened cytokine production in response to TLR4 activation with lipopolysaccharide. However, the response to TLR2 stimulation with peptidoglycan was unchanged in the ESRD group. Conclusions: Monocyte TLR2 and TLR4 and neutrophil TLR4 expressions and TLR4 activity are increased hemodialysis patients, representing another dimension of ESRD-associated inflammation.