Darla S. Morton

The Acute Response of the Immune System to Tennis Drills in Adolescent Athletes

any components of the immune system exhibit M change after intensive and prolonged exertion, including neutrophilia (high blood neutrophil counts), lymphopenia (low blood lymphocyte counts), decrease in natural killer cell cytotoxic activity, decrease in mitogeninduced lymphocyte proliferation (a measure of T cell function), increase in plasma concentrations of proand anti-inflammatory cytokines, for example, interleukin-6 ( I M ) , interleukin-10 (ILlO), and interleukin-1 recep tor antagonist (ILlra) , decrease in ex vivo production of cytokines in response to mitogens, and decrease in nasal and salivary IgA concentration (see reviews by Gabriel & Kindermann, 1997; Mackinnon, 1999; Nieman, 1997; Nieman & NehlsenCannarella, 1994). Depending on the immune measure, these changes take from 3 hr to 3 days to return to pre-exercise levels. Together, these data suggest that the immune system is suppressed and stressed, albeit transiently, following prolonged endurance exercise.

Effects of Social Conflict on Immune Responses and E. coli Growth Within Closed Chambers in Mice

Social conflict has been shown to affect the neuroendocrine stress response in rodents. The current study was designed to characterize the effects of social conflict on leukocyte subset distribution and function as well as in vivo bacterial growth. Male DBA/2 mice implanted or not implanted with a closed chamber containing Escherichia coli were repeatedly challenged by temporary placement in the territory of a dominant CF-1 mouse five times a day for 2 consecutive days. Nonstressed animals were similarly handled, but were not exposed to social conflict. Effects on immune responses and E. coli growth were analyzed 13 h after the last social conflict session. Social conflict alone was associated with an increase in plasma corticosterone concentration and decreases in thymocyte numbers and splenocyte ability to proliferate in vitro in the presence of lipopolysaccharide (p < 0.05). After social conflict, immature CD4+CD8+ thymocytes decreased, whereas mature T cells increased (p < 0.05). In the presence of E. coli, social conflict induced a significant increase in plasma concentration of interleukin-1beta, and a decrease in the number of thymocytes and the percentage of CD4+CD8+ T cells in the thymus (p < 0.05). In addition to the lymphocyte subpopulation changes observed with social conflict alone, the proportion of CD3+ and major histocompatibility complex (MHC) class II IAd+ cells were significantly higher in stressed mice implanted with a closed chamber containing E. coli (p < 0.05). Social conflict tended to favor E. coli growth in the closed chamber, indicating possible direct bacterial-neuroendocrine hormone interactions. Taken together, these results suggest that stress may modulate the host immune response by altering both bacterial growth and resistance to infection.

Inhibition of Interferon, Cytokine, and Lymphocyte Proliferative Responses in Elite Swimmers with Altitude Exposure

To determine the immunologic consequences of athletic training at altitude, blood samples were taken at rest from 10 swimmers and 8 control nontraining but altitude-exposed members of the 1996 Australian Olympic Swimming Team, near the start and completion of a 21-day training camp at 2102 m. Blood leukocyte numbers dropped in both groups (p < 0.05), with the decrease greater in the swimmers (-38% swimmers, -3% controls). Concanavalin A (ConA)-induced blastogenesis decreased in both groups (p < 0.01), but the drop was greater in the control group (-32% swimmers, -56% controls, p < 0.05). Lipopolysaccharide (LPS)-induced blastogenesis more than doubled in both groups (281% swimmers, 249% controls, p < 0.01). Increases in mitogen-induced interleukin-1beta (IL-1beta), IL-4, and interferon-gamma (IFN-gamma) production and a decrease in IL-2 levels were observed in both groups after altitude exposure (all p < 0.05). The percentage of cells expressing HLA-DR fell (-33% swimmers, -20% controls, p < 0.01), whereas those expressing CD-4 expression increased (16% swimmers only, p < 0.01). Although training at medium-level altitude alters some immunologic parameters, the training-induced changes may be secondary to those induced by altitude alone.

Effects of 2-deoxy-D-glucose administration on immune parameters in mice.

Physical exercise and diet alterations have been shown to affect immune parameters. Similar effects are also induced by the administration of the non-metabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The current study was designed to characterize the effects of glucoprivation induced by 2-DG administration on leukocyte subset distribution and function. BDF1 mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, 1000 or 1500 mg/kg of 2-DG. Two hours after the last injection of 2-DG, immunological parameters were analyzed. A dose-dependent increase in plasma glucose concentrations of mice injected once with up to 1500 mg/kg of 2-DG was observed (p < 0.001). After either one or three injections of up to 1500 mg/kg of 2-DG, corticosterone levels, leukocyte counts in the spleen, and CD3+ cells in the thymus increased. In vitro proliferation of partially purified lymphocytes from the spleen in the presence of both concanavalin-A and lipopolysaccharide decreased in a dose dependent manner (p < 0.05). In addition, after three injections, the proportion of both thymocytes and splenocytes bearing alphabeta-TCR increased as the concentration of 2-DG increased (p < 0.01). These results demonstrate that 2-DG administration induced dose-dependent changes in both thymus and spleen cell distribution and function.

Human annulus cells regulate PAPP-A and IGFBP-4 expression, and thereby insulin-like growth factor bioavailability, in response to proinflammatory cytokine exposure in vitro

Abstract Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase which cleaves IGF binding protein (BP)-4 in the extracellular matrix, making IGF available to nearby cells. We have shown that PAPP-A is present in the human intervertebral disc, and is significantly upregulated in more degenerated discs where increased proinflammatory cytokine levels are present. We hypothesized that increased proinflammatory cytokines present in the degenerating disc might be related to PAPP-A expression. Experiments exposed human annulus cells to IL-1-β or TNF-α to test this hypothesis. Treated cells showed significantly increased PAPP-A in conditioned media versus controls (p < 0.001). PAPP-A production following exposure to IL-1β was significantly greater in cells derived from more degenerated versus healthier discs (p = 0.05). PAPP-A gene expression (microarray analysis) was significantly upregulated in IL-1β- or TNF-α-exposed cells (p = 0.01–0.004). Quantitative RT-PCR confirmed significant upregulation of IGFBP-4 in IL-1β- or TNF-α-exposed cells. Data have potential relevance to future cell-based biologic therapies for disc degeneration.

Immune Alterations in Male and Female Mice after 2-Deoxy-d-Glucose Administration

Administration of 2-deoxy-D-glucose (2-DG) induces acute cellular glucoprivation. In the current study, we examined differences in immune parameters after 2-DG administration in both sexes. Male and female BDF1 mice were injected three times, 48 h apart, either with a saline solution (control group) or with 2-DG in saline (500 mg/kg). Two hours after the last injection, blood and spleens were collected. Plasma levels of interleukin-1beta, and interferon-gamma levels were measured. Additionally, the levels of the specific leukocyte antigens CD3, CD4, CD8, T cell receptor (TCR) alpha/beta, I-Ad, and H-2Ld/H-2Db were evaluated by flow cytometry on both blood and spleen cells. The blastogenic response of leukocytes from both tissues to mitogens was assessed. Levels of glucose, corticosterone, testosterone, progesterone, 17beta-estradiol, follicle-stimulating hormone, and luteinizing hormone were also determined. Increases in the percentage of cells bearing TCR alpha/beta and I-Ad in the blood and H-2Ld/H-2Db in the spleen were observed in the 2-DG-treated group for both sexes. In contrast, higher corticosterone and IL-1beta plasma concentrations, as well as higher percentages of splenocytes bearing TCR alpha/beta and I-Ad, and lower mitogen-induced proliferation of mature T splenocytes (79%) were observed in female but not in male mice injected with 2-DG compared with those injected with saline (p < 0.05). Taken together, these results suggest that female mice are more sensitive than male mice to immune alterations induced by 2-DG administration.

Effects of 2-deoxy-D-glucose administration on cytokine production in BDF1 mice.

Physical exercise and diet changes have been shown to affect immune parameters, and similar effects are also induced by the administration of a nonmetabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The present study was designed to characterize the effects of glucoprivation induced by 2-DG administration on concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in the blood and interferon-gamma (IFN-gamma), IL-2, and IL-4 in vitro production by partially purified T splenocytes in BDF1 mice. Mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, or 1000 mg/kg of 2-DG, and blood and spleens were collected 2 h after the last injection. Partially purified T splenocytes were cultured 24 h in the presence of concanavalin A (ConA). A significant increase in the corticosterone levels with the amount of 2-DG injected was observed after one or three injections (p<0.05). The amount of 2-DG injected was associated with an increase in TNF-alpha, IL-1beta, and IL-6 concentrations in the blood of mice after one or three injections of 2-DG (p<0.05). A significant decrease in in vitro proliferation of partially purified splenocytes in the presence of ConA was associated with a decrease in IFN-gamma production in the culture supernatants and an increase in IL-1 receptor expression on the cell surface (p<0.05).

Immune alterations in three mouse strains following 2-deoxy-d-glucose administration

Using 2-deoxy-D-glucose (2-DG)-induced stress, our laboratory has developed studies to define stress effects on immune responses. Here, we report effects of increasing doses of 2-DG on the immune response of BALB/c, C57BL/6 and BDF(1) mice 2 h after three injections of 0 to 2000 mg/kg of 2-DG. Female 4- to 5-week-old mice were euthanized and blood and spleens were collected. A suspension of partially purified mature T splenocytes was obtained by negative selection using J11.d2 antibodies. Glucose and corticosterone levels were measured in the plasma of each mouse. Splenocyte and mature T splenocyte suspensions were tested in in vitro proliferation assays with or without concanavalin A. Splenocytes were analyzed for the following cell-surface markers: CD3, TCR alpha/beta, CD4, CD8 and major histocompatibility complex (MHC) Class II. Significant increases in blood glucose levels were observed in C57BL/6 and BALB/c strains with the highest 2-DG dose (p<0.05). Corticosterone levels were higher in BDF(1) mice and C57BL/6 mice following the administration of 1000 and 2000 mg/kg of 2-DG, respectively (p<0.01). In vitro proliferation of mature T splenocytes in the presence of concanavalin A was decreased in BDF(1) (p<0.05) but not in BALB/c and C57BL/6 mice. In addition, in BDF(1) mice the decrease was highly correlated with an increase of CD3+ and TCR alpha/beta+ cells in the spleen. These results demonstrated high variability in the response of different mouse strains to 2-DG-induced stress.

Efficacy and Safety of Orally/Sublingually, Intranasally, and Intraperitoneally Administered Recombinant Murine Interferon in the Treatment of Murine Encephalomyocarditis Virus

Interferons (IFN) have been shown to be effective in protecting animals against lethal viral infections when administered systemically in relatively high doses. Intraperitoneal (i.p.) injection of mice with encephalomyocarditis virus (EMCV) gives rise to a rapidly progressive fatal disease characterized by central nervous system involvement and encephalitis. IFN-alpha has been shown to be effective in protecting mice against lethal EMCV infection when given via parenteral and oral/sublingual routes. The current study was designed to explore the ability of orally/sublingually and intranasally (i.n.) administered IFN-alpha to treat mice infected with EMCV in support of a planned clinical trial to evaluate efficacy of oral IFN-alpha in human viral infections. The primary objective of the study was to determine the efficacy of recombinant murine IFN-alpha (rMuIFN-alpha) in the treatment of mice infected with 100 LD(50) EMCV following oral, i.n., and i.p. administration at doses of 20,000 and 100,000 IU. The results of the current experiment did not indicate protection from infection with EMCV in mice that received IFN by the i.n. or oral/sublingual routes. The negative controls, infection of mice with 100 LD(50) of EMCV followed by treatment with excipient via all three routes, resulted in death of nearly all mice, as expected. The positive control, treatment of EMCV-infected (100 LD(50)) mice with rMuIFN-alpha via the i.p. route, was successful in protecting a significant number of mice from death compared with matched controls. This study points out the need to determine the optimum conditions for administration of oral/sublingual or i.n. IFN to insure maximum efficacy against viral infections.