The NPY effects on murine leukocyte adherence and chemotaxis change with age. Adherent cell implication

The two-way communication between the nervous and immune system is currently well-known, but the age-related changes in this communication have been scarcely studied. In the present work, we have investigated the in vitro effects of neuropeptide Y (NPY) at concentrations ranging from 10(-13) to 10(-7) M on the adherence and chemotaxis capacities of spleen, axillary node, thymus and peritoneum leukocytes from BALB/c mice. The NPY effect on these functions was examined on cells from animals of four different ages, i.e. young (12+/-2 weeks old), adult (24+/-2 weeks old), mature (50+/-2 weeks old) and old (72+/-2 weeks old). In young animals, NPY stimulates the adherence of leukocytes from spleen, axillary nodes and thymus and inhibits it in cells from peritoneum. In adult animals NPY inhibits the adherence of leukocytes from thymus. These effects disappear with ageing in all locations. Chemotaxis is stimulated by this neuropeptide at all ages in cells from axillary nodes and peritoneum, but this effect is absent in old mice. NPY exerts an inhibitory effect on the chemotaxis of leukocytes from thymus at all ages studied. These NPY effects on leukocytes seem to be carried out through adherent cells.     

Deterioration of cellular immunity during aging. The relationship between age-dependent impairment of delayed-type hypersensitivity reactivity, interleukin-2 production capacity, and frequency of Thy-1+,Lyt-2- cells in C57BL/Ka and CBA/Rij mice

The effect of aging on the delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in vivo and the interleukin-2 (IL-2) production capacity in vitro by spleen cells from young (17 weeks) and old (125 weeks) CBA/Rij and C57BL/Ka mice were investigated. For both CBA/Rij and C57BL/Ka mice an age-related decline in the DTH response to SRBC and the IL-2 production capacity was observed. Both parameters are mediated by Thy-1+,Lyt-2- spleen cells. For both mouse strains the proportion of Thy-1+,Lyt-2- spleen cells declined less strongly with aging than the DTH reactivity and the IL-2 production capacity. From this it was concluded that not only a quantitative but also a qualitative decrease of T-cell function occurs during senescence. It was also investigated whether the proportion of Thy-1+,Lyt-2- peripheral blood lymphocytes can be used as a predictive value with regard to the decline of DTH with aging of the corresponding mouse. This was indeed found to be the case in CBA/Rij mice, but not in C57BL mice.     

Splenic and inguinal lymph node T cells of aged mice respond differently to polyclonal and antigen-specific stimuli.

Numerous changes have been reported to occur in T cell responsiveness of mice with increasing age. However, most of these studies have examined polyclonal stimulation of spleen cells from a limited number of mouse strains. This study investigated the influence of genetic background, source of lymphocytes, and type of stimulus on age-associated changes in T cells response. Con A-induced proliferation and IL-2 and IFN-gamma production by splenic lymphocytes (SL) was significantly greater in CBA/Ca mice compared to C57BL/6 mice, regardless of age. SL of both strains exhibited the predicted age-dependent decline in proliferative response and an increase in IFN-gamma production in response to Con A. In contrast, however, only SL from C57BL/6 mice demonstrated the predicted age-dependent decline in Con A-induced IL-2 production; Con A-induced SL of young and aged CBA/Ca mice produced comparable amounts of IL-2. Differences in age-associated responses to Con A were also observed between SL and inguinal lymph node (ILN) cells of CBA/Ca mice. In contrast to SL, ILN cells demonstrated an increased proliferative response to Con A. However, lymphokine production by Con A-stimulated ILN cells from aged CBA/Ca mice was similar to that of Con A-stimulated SL from aged CBA/Ca mice. To determine if aged ILN T cells respond similarly to polyclonal and antigen-specific stimuli, keyhole limpet hemocyanin (KLH) responses of T cells isolated from ILN of aged and young CBA/Ca mice were examined. KLH-specific T cells from aged mice cultured with KLH-pulsed macrophages (M phi) from aged mice were significantly reduced in their ability to proliferate compared to KLH-specific T cells of young mice cultured with young KLH-pulsed M phi. In contrast to the expected results, the defect was not at the level of the T cells; proliferation of young T cells cultured with aged KLH-pulsed M phi was equivalent to the proliferation of aged T cells cultured with aged M phi. These results suggest that aging has differential effects on polyclonal and antigen-specific T cell proliferation and on polyclonal stimulation of T cells isolated from different lymphoid organs and from different strains of mice.     

Neuropeptide Y cotransmission with norepinephrine in the sympathetic nerve-macrophage interplay

The CNS modulates immune cells by direct synaptic-like contacts in the brain and at peripheral sites, such as lymphoid organs. To study the nerve-macrophage communication, a superfusion method was used to investigate cotransmission of neuropeptide Y (NPY) with norepinephrine (NE), with interleukin (IL)-6 secretion used as the macrophage read-out parameter. Spleen tissue slices spontaneously released NE, NPY, and IL-6 leading to a superfusate concentration at 3-4 h of 1 nM:, 10 pM:, and 120 pg/ml, respectively. Under these conditions, NPY dose-dependently inhibited IL-6 secretion with a maximum effect at 10(-10) M: (p = 0.012) and 10(-9) M: (p < 0.001). Simultaneous addition of NPY at 10(-9) M: and the alpha-2-adrenergic agonist p-aminoclonidine further inhibited IL-6 secretion (p < 0.05). However, simultaneous administration of NPY at 10(-9) M: and the beta-adrenergic agonist isoproterenol at 10(-6) M: or NE at 10(-6) M: significantly increased IL-6 secretion (p < 0.005). To objectify these differential effects of NPY, electrical field stimulation of spleen slices was applied to release endogenous NPY and NE. Electrical field stimulation markedly reduced IL-6 secretion, which was attenuated by the NPY Y1 receptor antagonist BIBP 3226 (10(-7) M, p = 0.039; 10(-8) M, p = 0.035). This indicates that NPY increases the inhibitory effect of endogenous NE, which is mediated at low NE concentrations via alpha-adrenoceptors. Blockade of alpha-adrenoceptors attenuated electrically induced inhibition of IL-6 secretion (p < 0.001), which was dose-dependently abrogated by BIBP 3226. This indicates that under blockade of alpha-adrenoceptors endogenous NPY supports the stimulating effect of endogenous NE via beta-adrenoceptors. These experiments demonstrate the ambiguity of NPY, which functions as a cotransmitter of NE in the nerve-macrophage interplay.     

Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2

The effects in vitro of sulphated and desulphated cholecystokinin (CCK)-8, and of gastrin-17 and gastrin-34 were studied at concentrations from 10⁻¹⁴ M to 10⁻⁶ M on several functions of human peripheral blood lymphocytes, i.e.: adherence to substrate, mobility (spontaneous and directed by a chemical gradient or chemotaxis), and spontaneous and phytohaemagglutinin (PHA)-mediated proliferation. All peptides, at concentrations from 10⁻¹⁰ M to 10⁻⁸ M, inhibited significantly the mobility capacity and PHA-induced proliferation, and increased the adherence and spontaneous proliferation. A dose-response relationship was observed, with a maximum response of lymphocyte functions at 10⁻¹⁰ M. These peptides induced a significant increase of intracellular cAMP levels at 30 and 60 sec. Because lymphoproliferation requires production of interleukin 2 (IL-2) by lymphocytes, we also measured the IL-2 production in the presence of the CCK and gastrin peptides, finding that this production was higher than in the respective controls. When peptides were added to samples containing PHA, the IL-2 production was significantly decreased with respect to samples incubated with PHA alone. These results suggest that the CCK and gastrin peptides are negative modulators of lymphocyte mobility (spontaneous mobility and chemotaxis), causing an inhibition of these activities through an increase of intracellular cAMP levels, and of PHA-induced lymphoproliferation, which is mediated by a diminution of the IL-2 production by lymphocytes.     

Neuropeptide Y effects on murine natural killer activity: changes with ageing and cAMP involvement

Changes in the bidirectional interaction between the nervous and the immune systems have been proposed as a cause of ageing. Neuropeptides, such as neuropeptide Y (NPY), could show different effects on immune function with age. In the present work, we have studied the in vitro action of a wide range of NPY concentrations, i.e. from 10(-13) to 10(-7) M, on natural killer (NK) activity, a function which decreases with age. Spleen, axillary nodes, thymus and peritoneum leukocytes from mice of different ages: young (12+/-2 weeks), adult (24+/-2 weeks), mature (50+/-2 weeks) and old (72+/-2 weeks) were used. Stimulation by NPY of NK activity was observed in adult and mature animals in axillary nodes and thymus, and an inhibition in the spleen from young mice. The specificity of the NPY effect on cytotoxic activity was confirmed using a C-terminal fragment of NPY. Furthermore, cAMP levels in leukocytes were found to be decreased by NPY in adult mice, suggesting an involvement of this messenger system in the NK modulation by this neuropeptide.     

The early protective thymus-independent antibody response to foot-and-mouth disease virus is mediated by splenic CD9+ B lymphocytes

Infection of mice with cytopathic foot-and-mouth disease virus (FMDV) induces a rapid and specific thymus-independent (TI) neutralizing antibody response that promptly clears the virus. Herein, it is shown that FMDV-infected dendritic cells (DCs) directly stimulate splenic innate-like CD9(+) B lymphocytes to rapidly (3 days) produce neutralizing anti-FMDV immunoglobulin M antibodies without T-lymphocyte collaboration. In contrast, neither follicular (CD9(-)) B lymphocytes from the spleen nor B lymphocytes from lymph nodes efficiently respond to stimulation with FMDV-infected DCs. The production of these protective neutralizing antibodies is dependent on DC-derived interleukin-6 (IL-6) and on CD9(+) cell-derived IL-10 secretion. In comparison, DCs loaded with UV-inactivated FMDV are significantly less efficient in directly stimulating B lymphocytes to secrete TI antibodies. A critical role of the spleen in the early production of anti-FMDV antibodies in infected mice was also demonstrated in vivo. Indeed, either splenectomy or functional disruption of the marginal zone of the spleen delays and reduces the magnitude of the TI anti-FMDV antibody response in infected mice. Together, these results indicate that in addition to virus localization, the FMDV-mediated modulation of DC functionality is a key parameter that collaborates in the induction of a rapid and protective TI antibody response against this virus.     

Dietary phytosterols modulate T-helper immune response but do not induce apparent anti-inflammatory effects in a mouse model of acute, aseptic inflammation

Although most studies have focused on the cholesterol-lowering activity of phytosterols, other biological actions have been ascribed to these plant sterol compounds, one of which is a potential immune modulatory effect. To gain insight into this issue, we used a mouse model of acute, aseptic inflammation induced by a single subcutaneous turpentine injection. Hypercholesterolemic apolipoprotein E-deficient (apoE(-/-)) mice, fed with or without a 2% phytosterol supplement, were treated with turpentine or saline and euthanized 48 h later. No differences were observed in spleen lymphocyte subsets between phytosterol- and control-fed apoE(-/-) mice. However, cultured spleen lymphocytes of apoE(-/-) mice fed with phytosterols and treated with turpentine showed increased IL-2 and IFN-gamma secretion (T-helper type1, Th1 lymphocyte cytokines) compared with turpentine-treated, control-fed animals. In contrast, there was no change in Th2 cytokines IL-4 and IL-10. Phytosterols also inhibit intestinal cholesterol absorption in wild-type C57BL/6J mice but, in this case, without decreasing plasma cholesterol. Spleen lymphocytes of turpentine-treated C57BL/6J mice fed with phytosterols also showed increased IL-2 production, but IFN-gamma, IL-4 and IL-10 production was unchanged. The Th1/Th2 ratio was significantly increased both in phytosterol-fed apoE(-/-) and C57BL/6J mice. We conclude that phytosterols modulate the T-helper immune response in vivo, in part independently of their hypocholesterolemic effect in a setting of acute, aseptic inflammation. Further study of phytosterol effects on immune-based diseases characterized by an exacerbated Th2 response is thus of interest.     

Enhancement by various cytokines or 2-beta-mercaptoethanol of Ia antigen expression on Langerhans cells in skin from normal aged and young mice. Effect of cyclosporine A

The number of Ia+ Langerhans cells (LC) in skin from aged (16- to 18-mo old) BALB/c mice is approximately 40% lower than in skin from young (2- to 3-mo old) mice. Overnight incubation at 37 degrees C with a variety of cytokines, including IL-1, IL-2, IL-3, IL-4, IL-6, TNF-alpha, IFN-gamma, and granulocyte/macrophage-CSF, causes a significant increase in the Ia expression on LC and raises the number of Ia+ cells by at least 300/mm2 in skin from both young and old mice. At 1 to 5 x 10(-5) M, 2-ME has a similar effect. The percentage increment in Ia+ cells is much higher for skin from aged mice than from young mice, particularly with IL-3, which appears to reconstitute the number of Ia+ LC in skin from aged mice to that of IL-3 exposed skin from young mice. Under the incubation conditions of our experiments, neither keratinocytes nor Thy-1+ dendritic cells acquire Ia Ag. Addition of 10 micrograms/ml cyclosporine A to the medium abolishes the effect of 2-ME and of all of the cytokines except IL-2 and IL-6. These results demonstrate the presence of a significant population of Ia- LC in the epidermis of both young and aged mice. It is suggested that epidermal production of cytokines may be of importance in the maintenance of constitutive Ia expression on LC. The possible interaction between keratinocytes and LC and the effect of aging on this process are discussed.     

1, 25 Dihydroxyvitamin D3 modulated cytokine response in pulmonary tuberculosis

1, 25 Dihydroxyvitamin D(3) (1, 25(OH)(2) D(3)) has gained significant importance in tuberculosis with regard to its immunoregulatory activities. Our aim was to evaluate the effect of 1, 25(OH)(2) D(3) on cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 60 healthy controls and 52 pulmonary tuberculosis patients were cultured with culture filtrate antigen (CFA) of M. tuberculosis and live M. tuberculosis with and without 1, 25(OH)(2) D(3) (10(-9), 10(-8)and 10(-7)M concentrations). The culture supernatants were used to estimate IL-8, IL-6, TGF-beta, IL-10, IFN-gamma, IL-12p40, IL-2, IL-4 and IL-5 levels by ELISA. 1, 25 Dihydroxyvitamin D(3) significantly suppressed IL-12p40 and IFN-gamma production in response to CFA and live M. tuberculosis with a maximum suppression at 10(-7)M concentration (p<0.0001). In CFA stimulated cultures, addition of 1, 25(OH)(2) D(3) significantly suppressed IL-8, IL-6 and IL-10 whereas the IL-2 levels were significantly increased in controls. It variably influenced the Th2 cytokines, showing an increased trend for IL-4 and suppressed IL-5 levels. We report that 1, 25(OH)(2) D(3) differentially modulates production of cytokines in response to M. tuberculosis antigens by predominantly suppressing IL-12p40 and IFN-gamma production in a dose dependent manner. Our results suggest a role for vitamin D in restricting acquired immune response against tuberculosis by regulating cytokine production.     

Lymphokines and aging: interleukin-2 production and activity in aged animals

The capacity of aged animals to produce and respond to the T cell-replacing factor, interleukin-2 (IL-2), has been examined. IL-2 activity in the supernatants of concanavalin A-activated aged spleen cells is 5- to 10-fold lower than comparable supernatants prepared using young spleen cells. This lesion in IL-2 synthesis may limit antibody production to T-dependent antigens, because supplementation with purified IL-2 markedly enhances the number of anti-SRBC plaques generated by aged spleen cells. The response of aged splenocytes can be fully restored to that obtained using young adult cells. However, there appears to be a defect in the ability of aged cells to effectively translate the IL-2 signal into B cell helper activity, in the absence of T lymphocytes. That is, although young adult, nylon wool-purified T cells can interact with aged T-depleted spleen cells, producing a normal high level anti-SRBC response, IL-2 is incapable of reconstituting the response in aged animals to this level. On the other hand, both young adult T cells and IL-2 can interact with young adult T-depleted splenic lymphocytes to produce a normal, high level anti-SRBC response.     

Effects of dietary omega3 and omega6 lipids and vitamin E on proliferative response, lymphoid cell subsets, production of cytokines by spleen cells, and splenic protein levels for cytokines and oncogenes in MRL/MpJ-lpr/lpr mice

omega3 Fatty acid rich fish oil (FO) and vitamin E may delay the progress of certain autoimmune diseases. The present study examined the mechanisms of action of omega3 lipids and vitamin E in autoimmune-prone MRL/lpr mice suffering from extensive lymphoproliferation, lupus-like symptoms, and accelerated aging. To determine whether the effects of omega3 lipids in autoimmune disease is linked to vitamin E levels, weanling female MRL/lpr and congenic control MRL/++ mice were fed diets containing 10% corn oil (CO) or 10% FO at two levels of vitamin E (75 IU or 500 IU/kg diet) for 4 months. The appearance of lymph nodes was delayed in the mice fed FO, and higher levels of FO offered further protection against the appearance of lymph nodes. Analysis of the spleen cells revealed that the cells positive for Thy.1 and Fas were significantly higher in the MRL/++ mice. The groups fed high levels of vitamin E generally exhibited higher levels of Fas. The proliferative response of splenocytes of MRL/++ mice to mitogens was significantly higher compared with MRL/lpr mice. Interleukin (IL)-10 production by spleen cells was significantly higher in FO-fed MRL/lpr mice than in CO-fed mice. In mice fed a high level of vitamin E, the production of IL-12 and tumor necrosis factor-alpha was significantly lower and IL-2 was significantly higher than in animals fed a low level of vitamin E. Proinflammatory cytokines were higher in the MRL/lpr mice and both FO and vitamin E lowered the levels of proinflammatory cytokines and lipid mediators. Western blots revealed that c-myc and c-ras were significantly lower and IL-2 and transforming growth factor (TGF)-beta1 levels were significantly higher in the spleens of MRL/++ mice. FO lowered c-myc and high levels of vitamin E in the diets normalized the levels of TGF-beta1 in MRL/lpr mice. The observations from this study suggest that both FO and vitamin E modulate the levels of specific cytokines, decrease the levels of proinflammatory cytokines, inflammatory lipid mediators, and c-myc, and increase TGF-beta1 levels in spleens of MRL/lpr mice and thus may delay the progress of autoimmune diseases.     

IL-10 signaling is essential for 1,25-dihydroxyvitamin D3-mediated inhibition of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) results from an aberrant, neuroantigen-specific, T cell-mediated autoimmune response. Because MS prevalence and severity decrease sharply with increasing sunlight exposure, and sunlight supports vitamin D(3) synthesis, we proposed that vitamin D(3) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) may protect against MS. In support of this hypothesis, 1,25-(OH)(2)D(3) strongly inhibited experimental autoimmune encephalomyelitis (EAE). This inhibition required lymphocytes other than the encephalitogenic T cells. In this study, we tested the hypothesis that 1,25-(OH)(2)D(3) might inhibit EAE through the action of IL-10-producing regulatory lymphocytes. We report that vitamin D(3) and 1,25-(OH)(2)D(3) strongly inhibited myelin oligodendrocyte peptide (MOG(35-55))-induced EAE in C57BL/6 mice, but completely failed to inhibit EAE in mice with a disrupted IL-10 or IL-10R gene. Thus, a functional IL-10-IL-10R pathway was essential for 1,25-(OH)(2)D(3) to inhibit EAE. The 1,25-(OH)(2)D(3) also failed to inhibit EAE in reciprocal, mixed bone marrow chimeras constructed by transferring IL-10-deficient bone marrow into irradiated wild-type mice and vice versa. Thus, 1,25-(OH)(2)D(3) may be enhancing an anti-inflammatory loop involving hemopoietic cell-produced IL-10 acting on brain parenchymal cells and vice versa. If this interpretation is correct, and humans have a similar bidirectional IL-10-dependent loop, then an IL-10-IL-10R pathway defect could abrogate the anti-inflammatory and neuro-protective functions of sunlight and vitamin D(3). In this way, a genetic IL-10-IL-10R pathway defect could interact with an environmental risk factor, vitamin D(3) insufficiency, to increase MS risk and severity.     

Effects of IL-10 and age on IL-6, IL-1beta, and TNF-alpha responses in mouse skeletal and cardiac muscle to an acute inflammatory insult.

Exaggerated proinflammatory cytokine responses can be observed with aging, and reduced levels of the anti-inflammatory cytokine IL-10 may contribute to these responses. IL-10 can reduce IL-6, IL-1beta, and TNF-alpha expression in nonmuscle tissues; however, no studies have examined the combined effects of IL-10 and age on cytokine responses in skeletal and cardiac muscle. These experiments tested the hypothesis that the absence of IL-10, in vivo, is associated with greater IL-6, TNF-alpha, and IL-1beta responses to an inflammatory challenge in skeletal and cardiac muscle and that aging exaggerates these responses. We compared IL-6, IL-1beta, and TNF-alpha mRNA and protein levels in skeletal and cardiac muscle of young (4 mo) and mature (10-11 mo) wild-type (IL-10(+/+)) and IL-10 deficient (IL-10(-/-)) mice following LPS. Skeletal and cardiac IL-6 mRNA and protein were elevated by LPS for IL-10(+/+) and IL-10(-/-) mice with greater responses in the IL-10(-/-) mice (P < 0.01). In skeletal muscle these effects were greater in mature than young mice (P < 0.01). IL-1beta mRNA and protein responses to LPS were greater in cardiac muscle of young but not mature IL-10(-/-) mice compared with IL-10(+/+) (P < 0.01). However, IL-1beta responses were greater in mature than young mice, but only in IL-10(+/+) groups (P < 0.05). The absence of IL-10 was associated with higher TNF-alpha protein levels in cardiac muscle (P < 0.05). The results provide the first in vivo evidence that the absence of IL-10 is associated with a greater IL-6 response to LPS in skeletal and cardiac muscles, and in skeletal muscle aging further exaggerates these responses.     

In vivo and in vitro synergistic antitumor effect of interleukin-2-cultured tumor-bearer spleen cells and immune fresh spleen cells

Summary The synergistic antitumor effect of interleukin-2(IL-2)-cultured tumor-bearer spleen cells (cultured lymphocytes) and immune fresh spleen cells was examined. Tumor-bearer cultured lymphocytes were obtained by culturing BALB/c spleen cells from syngeneic MOPC104E-tumor-bearing mice for 11 days with crude IL-2 and a soluble tumor extract. These cultured lymphocytes had weak antitumor activity when transferred i.p. into tumor-bearing mice that had been inoculated i.p. with 10⁵ tumor cells 5 days previously. Immune fresh spleen cells, obtained from mice in complete remission after the treatment with cyclophosphamide, also had weak antitumor activity when transferred at the same schedule. The cultured cells and the fresh cells, mixed together before transfer, significantly augmented the therapeutic effect. At least 1×10⁷ tumor-bearer cultured lymphocytes and 4×10⁷ immune cells were needed for the synergistic effect. A tumor-specific combination was needed for both cultured and fresh cells. The effective subpopulation of tumor-bearer cultured lymphocytes was a cytotoxic one from an Lyt2⁺ precursor, and that of the immune fresh spleen cells was noncytotoxic, Lytl⁺ and Lyt2⁺ T-cells.A similar synergistic effect was also observed during in vitro coculture of tumor-bearer and immune cells. Cytotoxicity, as assessed by the ⁵¹Cr-release test, of tumor-bearer IL-2-cultured lymphocytes was maintained most effectively after 3 or 4 days of culture without IL-2 when the lymphocytes were cocultured with immune fresh spleen cells and tumor cells.     

Influence of aging on murine neutrophil and macrophage function against Candida albicans

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.     

Il-10 is a central regulator of cyclooxygenase-2 expression and prostaglandin production

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10(-/-) mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10(-/-) mice. LPS-induced PGE(2) production from IL-10(-/-) spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10(-/-) spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10(-/-) mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10(-/-) mice. Neutralization of IFN-gamma, TNF-alpha, or IL-12 markedly decreased the induction of COX-2 in IL-10(-/-) spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10(-/-) mice. Treatment of IL-10(-/-) mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10's central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.     

Effect of age on homeostasis of lymphocytes in an interleukin-7-deficient mouse model

Interleukin (IL)-7 is thought to be a non-redundant cytokine for lymphopoiesis as there is a reduction of T and B cells in peripheral blood (PB) and a loss of TCRγδ+ cells in PB and bone marrow (BM) in IL-7?/? mice. To investigate whether the absence of IL-7 influences the organ-dependent distribution of the lymphocytes, we analyzed single cell suspensions of several organs (BM, lung, liver, small intestine, and spleen) at different ages (three and 12 months) of IL-7+/+ and IL-7?/? mice using flow cytometry; immunohistochemical staining was performed on frozen sections of various organs. We observed lymphocytopenia in almost all organs of IL-7?/? mice, but normal counts in the liver and the lung of three-month-old IL-7?/? mice. CD4+ and CD8+ cell numbers were decreased in the spleen and the BM. With aging, we found a greater increase in CD4+ and CD8+ cells in the BM of IL-7?/? than in IL-7+/+ mice, particularly of memory cells. The spleen of IL-7?/? mice was characterized by lymphocytopenia. We challenge the view that IL-7 is a non-redundant cytokine for lymphocyte development. Some of the changes observed, e.g. partial absence of TCRγδ+ T cells in the PB, BM and small intestine and complete loss in liver, lung and spleen, may be due to the altered organ distribution instead of a defect in γδ+ T cell lymphopoiesis. In this model, aging leads to a significantly altered composition of lymphocyte subsets, and the lack of IL-7 seems to accelerate this process.     

B cells moderate inflammatory progression and enhance bacterial containment upon pulmonary challenge with Mycobacterium tuberculosis

Though much is known about the function of T lymphocytes in the adaptive immune response against Mycobacterium tuberculosis, comparably little is understood regarding the corresponding role of B lymphocytes. Indicating B cells as components of lymphoid neogenesis during pulmonary tuberculosis, we have identified ectopic germinal centers (GCs) in the lungs of infected mice. B cells in these pulmonary lymphoid aggregates express peanut agglutinin and GL7, two markers of GC B cells, as well as CXCR5, and migrate in response to the lymphoid-associated chemokine CXCL13 ex vivo. CXCL13 is negatively regulated by the presence of B cells, as its production is elevated in lungs of B cell-deficient (B cell(-/-)) mice. Upon aerosol with 100 CFU of M. tuberculosis Erdman, B cell(-/-) mice have exacerbated immunopathology corresponding with elevated pulmonary recruitment of neutrophils. Infected B cell(-/-) mice show increased production of IL-10 in the lungs, whereas IFN-gamma, TNF-alpha, and IL-10R remain unchanged from wild type. B cell(-/-) mice have enhanced susceptibility to infection when aerogenically challenged with 300 CFU of M. tuberculosis corresponding with elevated bacterial burden in the lungs but not in the spleen or liver. Adoptive transfer of B cells complements the phenotypes of B cell(-/-) mice, confirming a role for B cells in both modulation of the host response and optimal containment of the tubercle bacillus. As components of ectopic GCs, moderators of inflammatory progression, and enhancers of local immunity against bacterial challenge, B cells may have a greater role in the host defense against M. tuberculosis than previously thought.     

Induction of natural killer cell activity and perforin and granzyme B gene expression following continuous culture of short pulse with interleukin-12 in young and old mice

Anticancer immunotherapy with cytokines is often limited by the occurrence of severe toxicity, particularly in older age groups, which are characterized by a reduced tolerance to antineoplastic therapies. We, and others, have recently demonstrated the efficacy of pulsing procedures with IL-2 as a new therapeutic strategy to induce antitumor cytotoxic cells. The aim of this paper was to evaluate the effect of IL-12 on NK cell activity in young and old mice and to investigate the possibility of inducing NK cytotoxicity and perforin and granzyme B gene expression through a brief exposure of spleen lymphocytes from young and old mice to IL-12. Pulsed lymphocytes were compared with non-pulsed cells cultured continuously in IL-12. IL-12 was able to boost both endogenous and IL-2-induced NK cell activity in young and old mice; the levels of cytotoxicity were lower in old than in young animals although the relative increase of IL-12 plus IL-2 versus IL-2 alone was greater for old mice. Comparable levels of NK cell activity were obtained in pulsed (5 min-1 hour) and non-pulsed lymphocytes from both young and old mice after one or three days of culture. The efficacy of the pulsing procedure was evident in both endogenous and IL-2-induced NK cytotoxicity. The mRNA encoding perforin and granzyme B were markedly and similarly enhanced in both IL-12-pulsed and non-pulsed lymphocytes in comparison with control cells. The results demonstrate the effectiveness of IL-12 pulsing in inducing antitumor cytotoxic cells, suggesting the possibility of using IL-12 pulsing, alone or in combination with IL-2, in the immunotherapy of both young and old subjects.