Christian R. Gomez

Serum from aged F344 rats conditions the activation of young macrophages

There is considerable controversy about the molecular mechanisms responsible for the variations in innate immunity associated with age. While in vivo, aged animals and humans react to an inflammatory signal with an excessive production of pro-inflammatory cytokines, studies in vitro generally show that this response is attenuated in macrophages from old individuals. In an effort to examine possible extrinsic factors that might affect the response of macrophages to lipopolysaccharide (LPS), we have challenged peritoneal macrophages obtained from young rats with sera obtained from rats of different ages. Our results indicate that the serum from aged rats significantly impairs the capacity of young macrophages to induce tumor necrosis factor-alpha (TNF-alpha) production, while at the same time it increases the basal levels of interleukin-6 (IL-6). The effect of serum from aged donors on TNF-alpha secretion requires pre-incubation and is sensitive to heat inactivation. In contrast, the stimulating effect on IL-6 is resistant to heat, and thus should not be due to a protein factor. Therefore, our results indicate that the age-related changes in macrophage activity are not only the consequence of intrinsic changes, but there also appears to be a modulatory effect imparted by the external milieu.

T-kininogen, a cystatin-like molecule, inhibits ERK-dependent lymphocyte proliferation

Plasma levels of kininogens increase with age in both rats and humans. Kininogens are inhibitors of cysteine proteinases, and filarial cysteine proteinase inhibitors (cystatins) reduce the proliferation of T cells. We evaluated whether T-kininogen (T-KG) might mimic this effect, and here we present data indicating that exposure of either rat splenocytes or Jurkat cells to purified T-KG results in inhibition of both ERK activation and [(3)H]-thymidine incorporation, both basal and in response to ConA or PHA. Interestingly, T-KG did not impair [(3)H]-thymidine incorporation in response to IL-2, which requires primarily the activation of the JNK and Jak/STAT pathways. These effects were neither the consequence of increased cell death, nor required the activity of kinin receptors. Furthermore, when T cell receptor proximal events were bypassed by the use of PMA plus Calcium ionophore, T-KG no longer inhibited ERK activation, suggesting that inhibition occurs upstream of these events, possibly at the level of membrane associated signal transduction molecules. We conclude that, like filarial cystatins, T-KG inhibits ERK-dependent T cell proliferation, and these observations suggest a possible role for T-KG in immunosenescence.

The release of sympathetic neurotransmitters is impaired in aged rats after an inflammatory stimulus. A possible link between cytokine production and sympathetic transmission

Aging results in a general decline in the response to external insults, including acute inflammatory challenges. In young animals, the inflammatory response requires activation of the sympathetic system, including neurotransmitters such as ATP, and catecholamines (epinephrine and norepinephrine). To test whether aging affects activation of this axis, and whether this in turn might affect cytokine release, we administered lipopolysaccharide (LPS) i.p. to adult, middle-aged and aged Fisher 344 rats (6-, 15- and 23-month old, respectively) and evaluated the early (0-12h) serum levels of Neuropeptide-Y (NP-Y), ATP and vanillyl mandelic acid (VMA, as an indirect measurement of catecholamine levels). In addition, we evaluated the association between these factors and serum levels of the cytokines tumor necrosis factor-alpha (TNFalpha) and interleukin-10 (IL-10). Induction of both ATP and NP-Y was markedly reduced in the serum of aged animals, when compared to their younger counterparts, while induction of VMA was not affected by age. In spite of these changes, serum levels of TNFalpha and IL-10 were strongly hyper induced and delayed in aged rats. The results suggest that during aging there is a dysregulation in sympathetic neurotransmitter regulatory mechanisms, and this might play a role in the impairment of the inflammatory response.

Bioelectrical Stimulation for the Reduction of Inflammation in Inflammatory Bowel Disease.

Crohns disease and ulcerative colitis are the primary inflammatory bowel diseases (IBDs) affecting the gastrointestinal tract. The current therapy aims at decreasing inflammation and reducing symptoms. This typically requires immune suppression by steroids, thiopurines, methotrexate, or tumor necrosis factor inhibitors. Patients may be unreceptive to medical therapy, and some may discontinue the treatment due to adverse effects. Noninvasive, transcutaneous vagus nerve stimulation (VNS) is currently used as a treatment for depression and epilepsy, and it is being investigated for the treatment of conditions such as multiple sclerosis, migraines, and Alzheimers disease. Recent studies have demonstrated the importance of splenic and vagus nerve functions in the inflammatory process through the production of certain cytokines. We hypothesize that using transcutaneous VNS via the auricular afferent branch could achieve a selective anti-inflammatory effect on the intestinal wall. This review examines the possibility of using vagal stimulators as a therapy for IBD. This could open the door to novel treatments for numerous vagally mediated diseases characterized by poor responses to current therapies.

T-kininogen induces endothelial cell proliferation

Basal proliferation of endothelial cells increases with age, and this might play a role in the etiology of age-related vascular diseases, as well as angiogenesis. Serum kininogen levels increase during aging in rats and humans, and T-kininogen (T-KG) can affect proliferative homeostasis in several cell models. Both kinins and kininogens have been shown previously to be angiogenic through activation of endothelial cell proliferation, and here we show that exposure of endothelial cells to T-KG results in vigorous cell proliferation, accompanied by ERK/AKT activation. In our experiments, the proliferative response requires B1 and B2 kinin receptors, even though kinins are not released from the precursor. We hypothesize that the age-related increase in T-KG could play a significant role in the age-related dysregulation of vascular physiology and function.

T-kininogen can either induce or inhibit proliferation in Balb/c 3T3 fibroblasts, depending on the route of administration.

T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition).