Christine Molinaro

Secondary necrosis is a source of proteolytically modified forms of specific intracellular autoantigens: Implications for systemic autoimmunity

OBJECTIVE Specific autoantigens targeted in systemic autoimmunity undergo posttranslational modifications, such as cleavage, during cell death that could potentially enhance their immunogenicity. In light of the increasing interest in the immunologic consequences of defective clearance of apoptotic cells, we sought to determine whether autoantigens cleaved during apoptosis undergo an additional wave of proteolysis as apoptosis progresses to secondary necrosis in the absence of phagocytosis. METHODS Apoptosis was induced in Jurkat cells with etoposide, anti-Fas antibody, or staurosporine (STS), and in HeLa cells with STS. Progression to secondary necrosis was assessed morphologically and quantified by trypan blue uptake. Autoantigen proteolysis during cell death was examined by immunoblotting of cell lysates using highly specific human autoantibodies as detecting probes. RESULTS Cells treated with the different apoptosis inducers underwent a rapid apoptosis that gradually progressed to secondary necrosis. During the initial apoptotic stages, several autoantigens, including poly(ADP-ribose) polymerase, topoisomerase I (or Scl-70), SSB/La, and U1-70 kd, were cleaved into their signature apoptotic fragments. Progression of apoptosis to secondary necrosis was associated with additional proteolysis of these and other autoantigens in a caspase-independent manner. Some autoantigens (e.g., ribosomal RNP, Ku, and SSA/Ro) appeared to be resistant to proteolysis during cell death. CONCLUSION In the absence of phagocytosis, apoptotic cells may undergo secondary necrosis, a process associated with additional proteolytic degradation of specific autoantigens. Secondary necrosis may occur in vivo in autoimmune disorders associated with impaired clearance of apoptotic cells and serve as a source of modified forms of specific autoantigens that might stimulate autoantibody responses under proinflammatory conditions.

Sympathetic innervation of the spleen in male Brown Norway rats: a longitudinal aging study.

Aging leads to reduced cellular immunity with consequent increased rates of infectious disease, cancer, and autoimmunity in the elderly. The sympathetic nervous system (SNS) modulates innate and adaptive immunity via innervation of lymphoid organs. In aged Fischer 344 (F344) rats, noradrenergic (NA) nerve density in secondary lymphoid organs declines, which may contribute to immunosenescence with aging. These studies suggest there is SNS involvement in age-induced immune dysregulation. The purpose of this study was to longitudinally characterize age-related change in sympathetic innervation of the spleen and sympathetic activity/tone in male Brown Norway (BN) rats, which live longer and have a strikingly different immune profile than F344 rats, the traditional animal model for aging research. Splenic sympathetic neurotransmission was evaluated between 8 and 32 months of age by assessing (1) NA nerve fiber density, (2) splenic norepinephrine (NE) concentration, and (3) circulating catecholamine levels after decapitation. We report a decline in NA nerve density in splenic white pulp (45%) at 15 months of age compared with 8-month-old (M) rats, which is followed by a much slower rate of decline between 24 and 32 months. Lower splenic NE concentrations between 15 and 32 months of age compared with 8M rats were consistent with morphometric findings. Circulating catecholamine levels after decapitation stress generally dropped with increasing age. These findings suggest there is a sympathetic-to-immune system dysregulation beginning at middle age. Given the unique T-helper-2 bias in BN rats, altered sympathetic-immune communication may be important for understanding the age-related rise in asthma and autoimmunity.

Sympathetic Nervous System and Lymphocyte Proliferation in the Fischer 344 Rat Spleen: A Longitudinal Study

Aging is associated with reduced cellular immunity, which leads to increased rates of infectious disease, cancer and autoimmunity in the elderly. Previous findings from our laboratory revealed an age-related decline in sympathetic innervation of immune organs that affects immunity. These studies suggested potential sympathetic nervous system involvement in age-induced immune dysregulation. Objectives: The purpose of this study was to longitudinally characterize the effects of age on sympathetic neurotransmission in the spleen and net sympathetic activity/tone in male Fischer 344 rats. Methods: Splenic sympathetic neurotransmission was evaluated between 8 and 24 months of age by (1) splenic norepinephrine (NE) concentration and turnover, (2) β-adrenergic receptor (β-AR) expression and (3) β-AR-stimulated splenocyte cAMP production. Measures of sympathetic neurotransmission were correlated with age-related changes in Concanavalin A (Con A)-stimulated splenocyte proliferation. Results: Splenic NE turnover increased during middle age, then subsequently declined by 18 months of age compared with 8-month-old controls (young). Splenic NE concentration increased at 10 months and decreased at 18–24 months, compared with young rats; however, plasma NE levels were not affected by age. Plasma epinephrine levels were decreased at 24 months. NE synthesis blockade increased and decreased the rate of plasma catecholamine depletion in middle and old age, respectively. β-AR-stimulated cAMP production increased in splenocytes by 15 months. An age-related decrease in Con A-induced splenocyte proliferation was apparent by 10 months and persisted through 24 months. The decline in Con A-induced splenocyte proliferation correlated with the age-related increase in cAMP production. Conclusions: Aging alters sympathetic nervous system metabolism in the spleen to affect β-AR signaling to splenocytes, suggesting that altered sympathetic-immune modulation changes are evident by early middle age.

Prevention of Mammary Tumor Development through Neuroimmunomodulation in the Spleen and Lymph Nodes of Old Female Sprague-Dawley Rats by L-Deprenyl

Background: Development of mammary tumors is an age-associated phenomenon that is likely due to deficits in the neuroendocrine-immune interactions. Previously, we demonstrated that L-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, can enhance immune responses and restore noradrenergic (NA) innervation in the spleens of rats with carcinogen-induced and spontaneously developing mammary tumors. Objectives: To investigate whether (1) treatment of early middle-aged female rats would prevent the spontaneous development of mammary tumors accompanied by restoration of immunity in the spleen and draining lymph nodes (DLN) and sympathetic NA innervation in the spleen and (2) deprenyl can influence the proliferation of estrogen receptor (ER)-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and Hs 578T) human breast cancer cells. Methods: Early middle-aged (8- to 9-month-old) female Sprague-Dawley rats were treated with 0, 1.0 or 2.5 mg of deprenyl/kg body weight (BW) daily i.p. for 12 months. Cells of ER-positive (ER+) and ER-negative (ER-) human breast cancer cell lines were incubated with media or 10-3 to 10-8M deprenyl for 1, 2, 4 or 6 days to examine the proliferation of cells. Results: Tumor incidence increased in saline-treated old female rats, while deprenyl treatment significantly reduced the incidence of mammary tumors in these rats. Saline-treated tumor-bearing rats exhibited reduced splenic NA innervation and norepinephrine (NE) content, splenic interleukin (IL)-2 and interferon (IFN)-γ levels and NK cell activity as well as DLN IL-2 and IFN-γ levels compared to young female rats without tumors. In contrast, treatment with 2.5 mg/kg of deprenyl enhanced IL-2 and IFN-γ production in both the spleen and DLN as well as splenic natural killer (NK) cell activity. Deprenyl treatment also increased concanavalin A (Con A)-induced proliferation of T lymphocytes in the DLN. Deprenyl-induced changes in immune responses were accompanied by enhanced NA innervation and NE content in the spleen. In vitro incubation of various concentrations of deprenyl with ER+ human breast cancer cell lines partly inhibited the proliferation of cells, while it had no effect on the ER- breast cancer cells. Conclusions: These results suggest that (1) development of mammary tumors is mediated through the loss of immunity and sympathetic NA nerve fibers accompanied by reduced NE levels in the spleen, (2) the prevention of mammary tumor development by deprenyl may involve the reversal of the tumor-associated decline in sympathetic NA activity and cell-mediated immune responses in the spleen and DLN and (3) the antitumor effects of deprenyl may be partially mediated through ER-dependent intracellular signaling pathways.

Chronically lowering sympathetic activity protects sympathetic nerves in spleens from aging F344 rats.

In the present study, we investigated how increased sympathetic tone during middle-age affects the splenic sympathetic neurotransmission. Fifteen-month-old (M) F344 rats received rilmenidine (0, 0.5 or 1.5mg/kg/day, i.p. for 90 days) to lower sympathetic tone. Controls for age were untreated 3 or 18M rats. We report that rilmenidine (1) reduced plasma and splenic norepinephrine concentrations and splenic norepinephrine turnover, and partially reversed the sympathetic nerve loss; and (2) increased β-adrenergic receptor (β-AR) density and β-AR-stimulated cAMP production. Collectively, these findings suggest a protective effect of lowering sympathetic tone on sympathetic nerve integrity, and enhanced sympathetic neurotransmission in secondary immune organs.

Chronically reducing sympathetic activity in the spleen during middle age does not reverse the age-related increase in β-AR-stimulated cAMP production in splenocytes

complications (r = .52, p = .041). Greater depressive symptomatology and greater total mood disturbance were also significantly associated with the development of wound healing complications at the surgical site during hospitalization (r = .53, p = .041 and r = .53, p = .043, respectively). Although drawn from a modest sample size, these data suggest PNI factors may be associated with clinically significant outcomes among women who are hospitalized secondary to TAH-BSO for endometrial cancer. Our ongoing/future research will identify the relevant biobehavioral confounds in this population, as well as assess the efficacy of presurgical psychological interventions on acute postsurgical outcomes in this population.

Sympathetic neurotransmission in spleens from aging Brown-Norway rats subjected to reduced sympathetic tone

Senescence of innate and adaptive responses and low-grade inflammation (inflammaging) hallmarks normal aging, which increases vulnerability to infectious diseases, autoimmunity and cancer. In normal aging, sympathetic dysregulation contributes to the dysregulation of innate and adaptive immunity and inflammaging. Sympathetic innervation of immune cells in secondary immune organs regulates immune responses. Previously in Fischer 344 (F344) rats, we reported an age-related increase in sympathetic tone and sympathetic dysfunction in beta-adrenergic receptor (AR) signaling of splenic lymphocytes that contributes to immune senescence, although the responsible mechanisms remains unexplored. In this study, we extend our previous findings using the much longer-lived Brown-Norway (BN) rats, whose behavior and immune response profile differ strikingly from F344 rats. Here, we investigated whether increased sympathetic nerve activity (SNA) in the aging spleen contributes to age-related sympathetic neuropathy and altered neurotransmission in splenic lymphocytes in BN rats. Fifteen-month male BN rats received 0, 0.5 or 1.5 μg/kg/day rilmenidine intraperitoneally for 90 days to lower sympathetic tone. Untreated young and age-matched rats controlled for effects of age. We found that elevated SNA in the aging BN rat spleen does not contribute significantly to sympathetic neuropathy or the aging-induced impairment of canonical β-AR signal transduction. Despite the rilmenidine-induced increase in β-AR expression, splenocyte c-AMP production was comparable with age-matched controls, thus dampening nerve activity had no effect on receptor coupling to adenylate cyclase. Understanding how aging affects neuroimmune regulation in healthy aging rodent models may eventually lead to strategies that improve health in aging populations vulnerable to immunosenescence and low-grade systemic inflammation.

173. Aging and stress effects on behavioral outcomes and tumor growth in a mouse model of prostate cancer

viral pathogens such as herpes simplex virus. While stress is thought to have a detrimental effect on the immune system’s ability to resist infection, studies have shown certain stressors (i.e., social disruption; SDR) can enhance innate immunity in the presence or absence of infection. Natural killer (NK) cells are bone marrow-derived innate lymphocytes known to be potent mediators in the early defense against viral challenges. Therefore, initial experiments exploring mechanisms by which SDR potentially enhances NK cell function in the absence of a pathogenic challenge were conducted. The objectives of these experiments were to determine the effect of an SDRinduced altered microenvironment on NK cell activation receptors (i.e., CD69 and CD16/32), MHC class 1-like inhibitory receptors (i.e., CD94/NKG2a and Ly49a), and cytolytic potential (i.e., CD107a). Flow cytometric analyses were used to examine the possible effects of SDR on NK cell receptor surface expression. Results indicate that SDR alone is capable of increasing surface expression of activating receptors, differentially reducing inhibitory receptor expression, and increasing cytolytic potential. Mechanisms found in these studies point to potential therapeutic targets in the prevention of viral infection, recurrence, severity, and transmission in susceptible patients.

135 EFFECTS OF AGING AND THE ANTIHYPERTENSIVE DRUG RILMENIDINE ON NERVE GROWTH FACTOR EXPRESSION IN THE SPLEEN OF MALE F344 RATS.

Previous research in our laboratory showed an age-related decline in noradrenergic (NA) sympathetic innervation of spleens from male Fischer 344 (F344) rats. Since nerve growth factor (NGF) plays an important role in determining the density of sympathetic innervation of target tissues throughout life, dysregulation of NGF expression in splenic target cells might explain the loss of sympathetic nerves. Moreover, chronically heightened sympathetic activity can also destroy nerve terminals by increased oxidative stress resulting from the breakdown of norepinephrine. The purpose of this study was to investigate whether these mechanisms are responsible for the age-related loss of splenic sympathetic nerves. Western blot analysis of NGF protein expression was evaluated in splenic lysates from untreated young (3 month) and old (18 month) male F344 rats and from 15-month-old rats treated with vehicle or rilmenidine (0.5 mg/kg and 1.5 mg/kg, bid [IP]) for 3 months to down-regulation of sympathetic activity. Our studies showed a 40% decrease in NGF protein levels in spleens from old rats compared with young adults. Noteworthy, rilmenidine induced a dose-dependent effect on NGF protein levels: administration of low and high doses of rilmenidine increased splenic NGF levels by 60 and 50%, respectively, compared with the vehicle group (sterile physiologic saline). Splenic NGF levels in the low-dose rilmenidine-treated group (500 μg/kg) were not different from those in 3-month-old F344 rats. Collectively, these findings support our hypothesis that changes in NGF protein expression might explain, at least in part, the age-related decline in sympathetic innervation of the spleen. Furthermore our data suggest that age-related changes in sympathetic activity in the spleen during middle age may precipitate the loss of NGF protein in spleens from old F344 male rats. This research is supported by NIH grant NS44032.

Effects of ageing and the antihypertensive drug rilmenidine on nerve growth factor (NGF) expression in the spleen of male F344 rats

ity to react to injury, might be of crucial importance as well. The purpose of this study was to investigate the changes in the expression of immune cells in the skin in relation to speed of wound healing and different psychological factors in humans. Twenty-two male non-smokers participated in this study. Every subject received a standard 4-mm punch biopsy. The healing process was monitored via ultrasound scanning, and the immune cells in the skin were analysed using different histological and immunohistochemical methods on the skin samples removed during the biopsy. Participants completed questionnaires on perceived stress, emotional distress, personality factors, and health behaviours 2 weeks prior, directly after and 2 weeks after the biopsy. The present study indicates the potential importance of the immune status of the skin as one of the possible pathways between psychological factors and wound healing. According to the present findings the level of activation of immune cells in the skin seems to be important in this process. Also, although in most of the analysis it failed to show statistically significant results, the expression of Langerhan’s cells deserves further investigation in this field. Macrophages showed no importance in this mechanism.