IKKß regulates endothelial thrombomodulin in a Klf2-dependent manner.

BACKGROUND: Endothelial thrombomodulin (TM) is critically involved in anticoagulation, anti-inflammation, cytoprotection and normal fetal development. Tumor necrosis factor alpha (TNFα) suppresses TM expression. OBJECTIVE: TNFα has been shown to down-regulate TM partly via activation of nuclear factor kappa B (NF-κB). However, because the TM promoter lacks an NF-κB binding site, the direct involvement of NF-κB has been controversial. We investigated the role of the upstream regulatory serine kinase, inhibitory kappa-B kinase-ß (IKKß), in TM expression and function with or without TNFα treatment. METHODS: Inhibition of IKKß was achieved by specific chemical inhibitors, siRNA or shRNA. TM expression was assessed by qRT-PCR, Western blot, flow cytometry, luciferase reporter assay and chromatin immune-precipitation (ChIP) assay. TM function was estimated by generation of activated protein C (APC). NF-κB activation was determined by immunocytochemistry. RESULTS AND CONCLUSIONS: IKKß inhibition increased TM expression and function, and attenuated TNFα-mediated TM down-regulation. In contrast, inhibition of downstream canonical NF-κB protein family members p50 and p65 (RelA) failed to up-regulate TM expression and did not affect IKKß inhibition-mediated TM over-expression. However, knockdown of cRel and RelB, family members of the canonical and non-canonical NF-κB pathway, respectively, resulted in TM over-expression. IKKß inhibition caused over-expression, increased promoter activity and enhanced binding of Krüppel-like factor 2 (Klf2) to the TM promoter, which positively regulates TM expression. Finally, knockdown of Klf2 completely attenuated IKKß inhibition-mediated TM up-regulation. We conclude that IKKß regulates TM in a Klf2-dependent manner.

Production of macrophage IL-1beta was inhibited both at the levels of transcription and maturation by caspase-1 following inhalation exposure to isobutyl nitrite.

Epidemiological studies have identified abuse of nitrite inhalants as an independent co-factor in HIV infection and in Kaposi's sarcoma (KS) in AIDS patients. In the present study we investigated the ability of macrophages from mice exposed to isobutyl nitrite to produce the inflammatory cytokine IL-1beta, upon stimulation with IFN-gamma and LPS. The production of IL-1beta was inhibited up to 55%. IL-1beta mRNA transcription was reduced by 35% following nitrite inhalant exposure, consistent with inhibition of activation-induced phosphorylation of macrophage mitogen-activated protein kinase p38. However, synthesis of the 31 kDa IL-1beta precursor protein was only marginally inhibited. Caspase-1, which cleaves the precursor IL-1beta into mature 17 kDa IL-1beta, was examined. Nitrite inhalant exposure blocked activation-induced increases in caspase-1 activity, consistent with a 50% reduction in 17 kDa IL-1beta shown in Western blots. Thus, exposure to nitrite inhalants reduced macrophage production of IL-1beta by reducing transcription, as well as post-translational processing mediated by caspase-1.

Inflammatory macrophage nuclear factor-kappaB and proteasome activity are inhibited following exposure to inhaled isobutyl nitrite.

A history of abuse of nitrite inhalants has been correlated with HIV seropositivity and Kaposi's sarcoma. A series of 14 daily, 45-min exposures of mice to 900-ppm isobutyl nitrite in an inhalation chamber reduced the number of peritoneal exudate macrophages (PEM) by 35% and the number of resident peritoneal macrophages (RPM) by 18%. Although the tumoricidal activity of RPM was not affected by the inhalant, the cytotoxicity of PEM was reduced by 26%. The induction of nitric oxide (NO) and the inducible NO synthase (iNOS) protein in PEM were inhibited by the inhalant to a similar extent. Inhibition of NF-kappaB activation in PEM from mice exposed to the inhalant corresponded to reduced degradation of the NF-kappaB inhibitor, IkappaB alpha. Proteasome-associated, enzymatic activity was compromised in PEM from inhalant-exposed mice, suggesting that inhaled isobutyl nitrite compromised macrophage, tumoricidal activity by inhibiting proteasomal degradation of the NF-kappaB inhibitor, IkappaB alpha.

Generation and characterization of a neutralizing monoclonal antibody against erythroid cell stimulating factor.

Erythroid cell stimulating factor (ESF) is present in mouse serum and has been reported to function in concert with erythropoietin (EPO) in the formation of erythroid cells in in vitro culture systems. We report here the generation and characterization of a monoclonal antibody (MAb) directed against ESF, with potent anti-ESF-neutralizing activity. A hybridoma-producing MAb to ESF was selected following enzyme-linked immunosorbent assay (ELISA)-based screening of 270 colonies obtained from a fusion of immunized mouse splenocytes with NS1 myeloma cells. Western blot analyses of mouse serum using this antibody specifically detected a single protein (approximate molecular weight of 60 kDa and 120 kDa, under reducing and nonreducing conditions, respectively) corresponding to ESF, with no reactivity to EPO. Furthermore, this MAb demonstrated reactivity to a protein similar in molecular mass, across species, showing reactivity in sera obtained from human, horse, goat, guinea pig, rabbit, and rat. Immuno-chemical characterization demonstrated this antibody to be of IgG3 isotype, bearing kappa light chains. Injection of this monoclonal anti-ESF antibody to exhypoxic polycythemic mice at 6 and 24 h after EPO injection significantly reduced 59Fe incorporation into red blood cells, demonstrating its ability to neutralize in vivo erythropoiesis in our mouse model system. Thus, this novel erythroid cell-specific MAb will be an invaluable tool for further delineating the physiological role of ESF in in vivo erythropoiesis.

Studies into the effect of tyrosine phosphatase inhibitor phenylarsine oxide on NFkappaB activation in T lymphocytes during aging: evidence for altered IkappaB-alpha phosphorylation and degradation.

Nuclear Factor kappa B (NFkappaB) is a critical regulator of several genes involved in immune and inflammatory responses. Treatment of T cells with a variety of stimuli, including TNF-alpha, leads to the translocation of the active p65-50 heterodimer to the nucleus, albeit at a lower level in T cells from the elderly. We demonstrate here that pretreatment with PAO results in the inhibition of NFkappaB induction in TNF-alpha treated T cells, suggesting a role for PAO-sensitive phosphatase in the activation of the NFkappaB via this pathway in human T cells. Furthermore, it demonstrates that aging does not influence the sensitivity of this phosphatase. Treatment with DMP prior to treatment with PAO and TNF abolishes the inhibition induced by PAO, in T cells from both young and old donors, alike. Finally, we demonstrate that a failure to degrade IkappaB-alpha in cytosols of TNF-treated T cells pretreated with PAO is due to its interference with the phosphorylation of IkappaB-alpha and not due to its inhibitory effect on proteasomal degradation. These data collectively suggest that PAO interferes with the phosphorylation and the regulated degradation of IkappaB-alpha, induced by TNF, without affecting the chymotryptic activity of the proteasome, independent of age.

Decreased proteasome-mediated degradation in T cells from the elderly: A role in immune senescence.

Induction of NFkappaB is a highly regulated process requiring phosphorylation, ubiquitination, and proteasome-mediated degradation of the cytosolic inhibitor IkappaBalpha. Analyses of the regulation of IkappaBalpha in TNF-alpha-treated T lymphocytes from young and elderly donors revealed severely compromised degradation of IkappaBalpha in T cells from the elderly. Examination of activation-induced phosphorylation and ubiquitination of IkappaBalpha did not demonstrate any significant age-related alterations. However, examination of proteasome activity in these T cells using fluorogenic peptide assays revealed a significant age-related decline in chymotryptic activity. These results suggest that a decline in proteasome activity results in a failure to fully degrade IkappaBalpha in the elderly. This failure to degrade IkappaBalpha may underlie both the observed decrease in NFkappaB induction and the IL-2 receptor expression in TNF-treated T cells during aging. Thus, decreased proteasome-mediated degradation may be central to immune dysfunction that accompanies aging.

Nuclear factor-kappaB induction in CD45RO+ and CD45RA+ T cell subsets during aging.

An increase in the ratio of memory to naive T cells has been postulated to underlie immune hyporesponsiveness accompanying aging. Our analyses of the induction of nuclear factor-kappaB (NFkappaB) in activated memory (CD45RO+) and naive (CD45RA+) T cell subsets from young and elderly donors has demonstrated that, regardless of donor age, memory T cells are not significantly altered in their responsiveness to TNF-alpha-mediated induction of NFkappaB. Although treatment with TNF-alpha induced nuclear localization of NFkappaB in both memory and naive T cell subsets, irrespective of the age of the donor, the levels of induced NFkappaB were significantly lower in both subsets of T cells obtained from the elderly, when compared to those in young. Examination of IkappaB alpha regulation revealed that TNF-alpha-mediated degradation of IkappaB alpha in both memory and naive T cells from the elderly was severely impaired, thus contributing to the lowered induction of the observed NFkappaB. In addition, this age-related decrease in induction of nuclear NFkappaB correlated with decrease in intracellular IL-2 receptor expression and anti-CD3-induced proliferation of both memory and naive T cells subsets. Taken together, our results suggest that the age-related hyporesponsiveness cannot be attributed to a skewing of the T cell population towards a memory phenotype in the elderly.

Decreased association of p56lck with CD4 may account for lowered tyrosine kinase activity in mitogen-activated human T lymphocytes during aging.

Treatment of T cells from young donors with PHA rapidly induces tyrosine phosphorylation of several intracellular substrates. In contrast, T cells from elderly donors treated in a similar manner showed far fewer tyrosine-phosphorylated proteins. To understand the basis of this age-associated difference in T cells, we examined the in vitro catalytic activity of kinase(s) following activation. We demonstrate both lowered overall in vitro kinase activity as well as a significant decrease in the activity of lymphocyte-specific tyrosine kinase p56lck in activated T cells from the elderly. Our results demonstrate for the first time an altered association of p56lck with coreceptors such as CD4 and CD45 in the elderly. These results suggest that alterations in p56lck tyrosine kinase and its association with CD4 and CD45 may underlie lowered T cell function during aging.

Regulation of transcription factor NF kappa B in immune senescence.

The Rel/NF kappa B family of eukaryotic transcription factors are critical in immune and inflammatory processes regulating the expression of a wide variety of cytokines including IL-1, IL-2, IL-6, TNF-alpha and GM-CSF. Its ubiquitous distribution, rapid induction and regulation, the complexity of its subunits and its apparent involvement in several diseases has made this transcription factor a subject of intense study in normal cellular growth and cancer. Emerging studies have implicated a role for this transcription factor in the normal processes of aging. As significant declines in immune function is a natural concomitant to advancing age, the regulation of transcription factor NF kappa B appears to play a pivotal role in immune dysregulation during senescence, contributing to down regulation of both IL-2 and IL-2 receptor-alpha expression. Our studies have contributed to understanding the regulation of lowered NF kappa B induction in T cells during aging in humans and mice. Since we have shown that the lowered induction of NF kappa B in activated T cells from the elderly can be attributed to impaired degradation of the inhibitor I kappa B-alpha due to lowered proteasomal activity, we suspect that a similar alteration in proteasomal activity may be operative in age-dependent failure of immune function including the inability to initiate DNA synthesis following activation, skewing of T cell repertoire, lowered cytolytic activity and accumulation of aberrant proteins. Understanding the regulation of the proteasome pathway during immune senescence may provide new avenues for therapeutic intervention for immune based geriatric diseases.

Excess formation of lysophosphatidic acid with age inhibits myristic acid-induced superoxide anion generation in intact human neutrophils.

A superoxide anion generation rate upon exposure to myristate of 1.93 +/- 0.34 nmol/min/10(6) cells in neutrophils from elderly human donors was significantly less than a value of 3.02 +/- 0.48 nmol/min/10(6) neutrophils from young donors. Myristate activation resulted in equal increases of AA in both the young and the old indicating no effect of aging on the PLA2 pathway to response. By contrast, the PLD-induced generation of PA was significantly higher in the old than in the young. In addition, myristate induced a significant age-related enhancement in LPA generation, in the old but not in the young. The mass of LPA generated following activation was 3.5 nmol/ 2.5 x 10(7) cells/ml in the young while in the old it averaged 7.0 nmol/2.5 x 10(7) cells/ml. The inhibitory effects of LPA may explain the age-related impaired ability to generate superoxide anion following activation by myristate.

Induction and regulation of NFkappaB during aging: role of protein kinases.

Aging is accompanied by significant declines in immune function. To understand the basis for this age-related decline we have investigated the induction and regulation of transcription factor NFkappaB in murine T cells. We report that anti-CD3 treatment of T lymphocytes from old mice resulted in significantly lower levels of NFkappaB than those observed in similarly treated T cells from young mice. Pretreatment of T cells with staurosporine reduced the induction of NFkappaB in anti-CD3-activated cells from young mice but had no effect in similarly treated T cells from old mice. However, pretreatment with H-89 had little or no effect on the induction of NFkappaB in T cells from young mice, but significantly enhanced the induction of NFkappaB in T cells from old mice. These results suggest that the impairment in the generation of NFkappaB during aging may reflect age-associated differences in the regulation of protein kinase A.

Evidence for lowered induction of nuclear factor kappa B in activated human T lymphocytes during aging.

Transcription factor NF kappa B (nuclear factor kappa B) is induced in T lymphocytes from young individuals following activation with a variety of stimuli including anti-CD3, phorbol myristate acetate (PMA), and tumor necrosis factor-alpha (TNF-alpha). In contrast, activated T lymphocytes from older individuals show a significant reduction in the induction of NF kappa B in response to the same stimuli. The age-related decline in induction of NF kappa B could not be attributed to alteration in the composition of subunits, p50 and p65 were found to be the predominant subunits of induced NF kappa B in T cells from young as well as elderly donors. Furthermore, similar levels of NF kappa B were found in the cytosols of unactivated T cells from both young and elderly donors suggesting that precursor levels of NF kappa B remain unaltered during aging. These results suggest that an age-associated decline in the induction of NF kappa B in activated T cells from elderly individuals may be attributable to altered regulation of the inhibitor, I kappa B, and may play an important role in immune dysregulation accompanying aging.

Effect of age on the fatty acid composition of phospholipids in human lymphocytes.

We have examined the fatty acid composition of phospholipids of unstimulated and PHA-stimulated T cells from young and old donors. Our results demonstrate that aging is accompanied by decreases in the saturated fatty acids, myristic acid, and palmitic acid, and a concomitant increase in the unsaturated arachidonic acid. Following activation with PHA for 24 h, age-associated differences in fatty acids could no longer be detected. In contrast to the lymphocyte, aging did not affect the fatty acid composition of either serum or neutrophil phospholipids. Exposure of lymphocytes from old donors to myristic acid complexed medium increased the levels of myristate in the phospholipids to levels similar to that seen in lymphocytes from young donors. We conclude from these studies that aging is accompanied by an alteration in the fatty acid profiles of phospholipids, and that incubation in myristic acid complexed medium modulates these profiles. These alterations are unique to lymphocytes and may contribute to the age-related declines in lymphocyte function.

Polymorphism of age-related changes in the antibody response to the hapten phosphorylcholine.

Aging is accompanied by changes in the immune system that occur at different levels and at different periods of time. We have studied age-related changes in isotype and idiotype of the antibody response to hapten phosphorylcholine (PC) in C57BL/6, and A mice and in the congenic MRL/Mp(-)+/+ and MRL/Mp-1pr/1pr strains. Three groups, representing young, middle and old age were immunized with PC-keyhole limpet hemocyanin. Total anti-PC antibody and the contribution of each isotype and of the T15 idiotype were assessed in the initial and late response. Some features of the antibody-response were similar in all the strains tested, e.g. the largest quantity of anti-PC antibody is formed in middle age and IgM is dominant in the initial response. However, remarkable differences occur in the isotype and idiotype predominance. Particularly, congenic MRL/Mp strains, prone to autoimmune disease, express the T15 idiotype only at low levels, even though IgM, which normally expresses this idiotype, is produced in large amounts. Furthermore, the late (memory) response of the MRL/Mp strains is dominated by IgG2b rather than IgG1, which is the predominant isotype in mice of long-lived strains. We conclude from these results that the number of T helper cells, involved in isotype regulation decreases with age and that there is a genetic variation, i.e., polymorphism in the ability to express T15-idiotype producing subtypes.

Fine specificity and isotype expression of anti-PC antibodies in BALB/c, MRL/Mp-lpr/lpr, and -(+)/+ mice at different ages.

The MRL/Mp congenic mouse strains develop autoimmune disease with age. We have investigated age- and autoimmune-related changes in fine specificity, isotype spectra and T15 idiotype expression of the anti-phosphorylcholine (PC) response in BALB/c, MRL/Mp- + and -lpr congenic mice and in (BALB/c x MRL/Mp-lpr) F1 hybrids. Two groups of anti-PC antibodies with distinct fine specificity are elicited in the memory response. Group I antibodies recognize the PC moiety and express the T15 idiotype. Antibodies of group II are specific for phenyl-phosphorylcholine and are found predominantly in the memory response. In the MRL/Mp-lpr and - + strains only a minor population of antibodies expresses the T15 idiotype at all ages. However, a third group of antibodies was observed which binds to PC-coated proteins and to Diplococcus pneumoniae R-36A. This binding was not inhibited by PC-chloride and appeared mainly in the memory response at old age. The isotype distribution among anti-PC antibodies was similar in all strains analysed. In the initial response primarily mu, gamma 3 and gamma 1 isotypes were produced, while in the memory response gamma 1 was dominant. Thus autoimmune defects and ageing result in altered anti-PC antibody and idiotype profile, probably related to altered states in both the T- and B-cell compartments.

Strain polymorphism in progression of aging: changes in CD4, CD8 bearing subpopulations.

Polymorphism of age-related changes in CD4 (L3T4) and CD8 (Lyt-2) determinants of spleen and thymus cells was assessed by fluorescence-activated flow cytometry. Cells from mice ranging from 5 weeks to greater than 2 years of age were examined. There is little age-related change in the proportion of CD4+ CD8- splenocytes in A, C57BL/6, DBA/1, DBA/2, and SJL mice (slopes 0.04, 0.06, 0.08, 0.17 and 0.17, respectively, when age in weeks was plotted against % of positive cells). Changes in the composition of the thymus are much more profound: CD4+ CD8+ cells of SJL mice decrease from 70% to less than 10% as the animals age from 5 to 69 weeks (slope -1.03), and in DBA/2 mice from 5 to 110 weeks (slope -0.88). While this decrease in CD4+ CD8+ cells occurs, there is a compensatory increase in CD4+ CD8- and CD4- CD8+ cells; this is a shift in the relative proportion of subpopulations rather than an increase in absolute cell numbers of a particular subpopulation. In contrast to the age-related changes of SJL and DBA/2 mice, there is relatively little change in the proportion of CD4+ CD8+ thymus cells in mice of strains C57BL/6, DBA/1 and A (slopes -0.03, -0.14 and -0.15, respectively).

Economic correction; quantity and age-related rate of change.

Correlation between the relative magnitude of activity or concentration in youth and the relative magnitude of age-related decrease occurs in several systems. We have observed this correlation in density of D2-dopamine receptors of the striatal membrane, activity of lymphocyte activated killer cells, relative density of CD8 on thymocytes, augmenting activity of T splenocytes and mRNA, coding for IL-1 in Langerhans cells. We have suggested that this correlation should be considered in the context of balanced investment in lifespan and reproductive efficiency, that it may be the result of feedback regulation and have designated it as "economic correction".

Immunosuppression after injection of Friend virus into C57BL/6 mice of different ages.

Injection with Friend virus (FV) causes immunosuppression in young and old C57BL/6 mice, i.e. it occurs whether or not the virus replicates very briefly or for a long period. There are only minor age-related differences in the extent of immunosuppression, except that suppression appears to persist somewhat longer in old than in young animals.

Age-dependent changes of isotype and idiotype expression in the antibody response to the phosphorylcholine hapten in BALB/c mice.

The distribution of antibodies among different isotypes in an immune response to a given antigen reflects the immunoregulatory linkage of T and B cell compartments, the genetic background of an individual and the functional state of its immune system. In addition, idiotypes are markers for the clonal origin of antibodies and their genetic relationship. Therefore, we have analyzed the isotype patterns and development of the major idiotype (T15) in BALB/c mice of different ages, immunized with a T-cell-dependent phosphorylcholine conjugate. The response was dominated by mu, gamma 3 and gamma 1 isotypes. The proportion of these antibodies changed in the progress of immunization but not with age in the primary response. An age-dependent change of the isotype distribution was observed only in the memory response. The T15 idiotype was dominantly expressed in the primary response and decreased in the memory response by 80-95% independently of the age of the mice. The results demonstrate that 2 populations of anti-phosphorylcholine antibodies which both prefer particular isotypes are expressed according to the functional state of the T and B cell compartments with age.

Effect of age on cutaneous interleukin 1 expression.

Age-related changes in epidermal immune function include morphologic and functional changes in Langerhans cells and decreased epidermal cytokine activity. In this study, expression of interleukin 1 (IL-1) mRNA was quantitated in 6-102-week old mice of different strains. A significant decline in cutaneous IL-1 expression occurred in mice of all strains examined; the rate of decline was most prominent in MRL/MpJ+ mice who also had highest initial levels of IL-1. This observation is in keeping with previous observations that the quantity of several gene products in youth is directly proportional to the rate of change of the quantity of that gene product. The term "economic correction" is used to describe this phenomenon.