|Year : 2019 | Volume
| Issue : 1 | Page : 33-37
Aging immunity, immunosenescence, or inflamm-aging: A comparative study of cytokines
Anand Gopal1, Dhiraj Kishore2, Inderjeet S Gambhir1, Amita Diwaker1
1 Department of Obstetrics and Gynecology, IMS, BHU, Varanasi, Uttar Pradesh, India
2 Department of Medicine, IMS, BHU, Varanasi, Uttar Pradesh, India
|Date of Web Publication||14-Oct-2019|
Department of Medicine, IMS, BHU, Varanasi, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Context: Aging has impact on immune system both quantitatively as well as qualitatively, leading to deregulated response in different states ranging from infection, autoimmunity, inflammation, and excessive tissue damage.
Aims: The present study is done to observe the immunosenescence-related changes in monocytes-/macrophages-mediated production of nitric oxide (NO), interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, anti-inflammatory cytokine IL-10 after stimulation with lipopolysaccharide (LPS), and interferon (IFN)-γ, evaluating the innate immunity of a group of healthy Indian elderly from a single institution.
Settings and Design: The study was conducted on life-term prisoners in Central Jail, Varanasi, India, and the Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, among the three groups, namely study group (>70 years old), control Group 1 (60–70 years), and control Group 2 (30–40 years).
Subjects and Methods: Mononuclear cells were separated, and NO production, pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α, and anti-inflammatory cytokine IL-10 from these cells were estimated by enzyme-linked immunosorbent assay after stimulation with LPS and IFN-γ.
Statistical Analysis Used: Mean and standard deviation were calculated, and Student's t-test was applied. Results were considered statistically significant forP < 0.05.
Results: A statistically significant rise in NO production (P < 0.001) was observed after stimulation as compared to control Groups 1 and 2 (P < 0.001). The study group showed a significant decrease in IL-1 production (P < 0.03) following stimulation. The basal TNF-α level was low in the study group, but there was a significant increase in TNF-α following stimulation. The basal level of IL-6 in the study group was higher than the control Group 1 (not significant, P = 0.21) but lower than the control Group 2 (not significant, P = 0.18). There were no significant inter- or intra-group changes in IL-6 level.
Conclusions: There is a qualitative as well as quantitative defect in elderly monocytes/macrophages reflecting abnormal inflammatory response. This could be due to abnormal immunity with aging, which may be responsible for increased frequency of chronic diseases such as diabetes, hypertension, congestive heart failure, asymptomatic infections, and Alzheimer's disease in geriatric population.
Keywords: Geriatrics, immunosenescence, innate immunity, monocytes
|How to cite this article:|
Gopal A, Kishore D, Gambhir IS, Diwaker A. Aging immunity, immunosenescence, or inflamm-aging: A comparative study of cytokines. J Med Soc 2019;33:33-7
|How to cite this URL:|
Gopal A, Kishore D, Gambhir IS, Diwaker A. Aging immunity, immunosenescence, or inflamm-aging: A comparative study of cytokines. J Med Soc [serial online] 2019 [cited 2020 Jan 26];33:33-7. Available from: http://www.jmedsoc.org/text.asp?2019/33/1/33/269106
| Introduction|| |
Immunosenescence is the decline in immune function that develops with age. The cumulative changes in critical B- and T-cell subpopulations and innate immune system lead to increased likelihood of infection, autoimmune diseases, and malignancy in elderly. A better understanding of immunosenescence may offer possible therapeutic intervention. Aged human shows several defects in monocytes/macrophages function. Hence, this comparative study was done on a group of healthy Indian elderly from a single institution to understand the alteration in innate immunity by the estimation of nitric oxide (NO) (nitric acid) production, pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α, and anti-inflammatory cytokine IL-10 by these cells after stimulation with lipopolysaccharide (LPS) and IFN-γ.
| Subjects and Methods|| |
The present study was done on life-term prisoners in Central Jail, Varanasi, and the Department of Medicine, Institute of Medical Sciences, Banaras Hindu University. The general criteria for selection in all groups were age, absence of diabetes, tuberculosis, or any other symptomatic infection or major disease on the basis of general examination and routine laboratory checkup. In all the three groups, namely study group (>70 years – 24 individuals), control Group 1 (60–70 years – 5 individuals), and control Group 2 (30–40 years – 4 individuals),in vitro function of monocytes (peripheral blood mononuclear cells [PBMC]) was studied for NO, IL-1, TNF-α, IL-6, and IL-10 production. Tissue culture media was purchased from Hyclone (Logan, Utah). Culture medium was supplemented with 20 μg/ml gentamycin, 100 μg/ml streptomycin, 100 IU penicillin, and 10% fetal calf serum (HiMedia, Mumbai). Histopaque-1077, used to separate mononuclear cells, and LPS were purchased from Sigma Diagnostics INC (St. Louis, USA); recombinant IFN-γ was obtained from NIBSC (Hertfordshire, UK). Antibodies against IL-1, IL-6, IL-10, and TNF-α were purchased from Imgenex (San Diego, CA). Secondary antibodies conjugated to alkaline phosphatase were obtained from Bengaluru Genic (Bengaluru). Heparinized blood (5 ml) was obtained from participants, layered over the same volume of histopaque-1077. It was then centrifuged at room temperature at 400 c/s for 30 min. The resulting band of mononuclear cells (PBMC) at interface was aspirated, washed twice in isotonic PBS, and resuspended in complete RPMI medium in mini Petri dish at a concentration of 106 cells/ml. LPS and IFN-γ were added at a concentration of 5 μg/ml and 51 U/ml, respectively, in all the three test groups. After 48 h of incubation in CO2 incubator at 37°C, the supernatant was aspirated. Cell lysate was prepared from mini Petri dish and the samples were stored at −20°C for future analysis. Nitrite production in the culture supernatant was determined by spectrophotometric assay method. Data were expressed as optical density (OD) at 550 nm. A standard enzyme-linked immunosorbent assay (ELISA) was performed to detect the presence of indicated cytokines, namely IL-1, IL-6, TNF-α, and IL-10. Absorbance was recorded at 405 nm in ELISA reader. Data were expressed as OD at 405 nm. Mean and standard deviation were calculated, and Student's t-test was applied. Results were considered statistically significant for P < 0.05.
| Results|| |
The mean age of the study group was 76.56 ± 2.76 years while that of the control Group 1 was 63.2 ± 2.05 years and control Group 2 was 34 ± 1.36 years. All the persons included in the study were male. Basal level of NO production was almost similar in three groups without significant difference; however, 48 h following activation, the study group showed a statistically significant rise in NO production (P < 0.001). There was a significant difference in the study group after stimulation as compared to the control Groups 1 and 2 (P < 0.001), but there was insignificant difference in the control Groups 1 and 2 (P = 0.33) [Table 1]. The basal level of IL-1 was higher in the study group than the control Group 1 (P = 0.009) and control Group 2 (P = 0.02). However, 48 h following activation, the study group showed a significant decrease in IL-1 production (P = 0.03), the control Group 1 also showed significant decrease (P < 0.001), but the control Group 2 showed insignificant increase in IL-1 production (P = 0.1) [Table 1]. The basal level of TNF-α was low in the study group (not significant, P = 0.07) and control Group 1 (not significant, P = 0.09) as compared to control Group 2. However, 48 h following activation, there was a significant increase in TNF-α in the study group, but both the control groups showed insignificant change [Table 1]. The basal level of IL-6 in the study group was higher than the control Group 1 (not significant, P = 0.21) but lower than the control Group 2 (not significant, P = 0.18). The basal level of IL-6 was also lower in the control Group 1 than the control Group 2 (P = 0.08, not significant). However, 48 h following activation, there were no significant inter- or intra-group changes in IL-6 level [Table 1]. The difference between basal level of IL-10 was not significant in the three groups, but 48 h following activation, the study group showed a tendency of higher IL-10 production (P > 0.05) while the two control groups showed a decrease in IL-10 production (P = 0.06 and P = 0.005, respectively). This increased level in the study group was not significantly higher than the control Group 1 (P = 0.2, not significant) and control Group 2 (P = 0.124, not significant) [Table 1].
|Table 1: Values of pre- and post-stimulation cytokines level with P value|
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| Discussion|| |
The present study was undertaken to study the pattern of immunosenescence afterin vitro stimulation of monocytes/macrophages (PBMC) with LPS and interferon (IFN)-γ. The monocytes of these three groups are more or less similar yield of PBMC and were studied for NO and cytokines production at basal levels and 48 h after stimulation. Results definitely showed altered immune response and interpreted as followings in context to variation in different markers. The basal level of NO was similar in all three groups; however, poststimulation, the study group showed a significant increase in NO production, but the control Group 2 showed a decline in NO production. NO is a unique molecule present in variety of cells and organs having multifaceted function; it is involved in a number of cellular signaling pathways. NO is synthesized through L-arginine oxidation by NO synthases (NOS). Inducible NOS gets expressed after exposure to stimuli such as IL-1, TNF-α, and LPS. However, transforming growth factor-β, IL-4, and IL-10 inhibit the inducible NO expression. Function of NO is concentration dependent. It helps in relaxation of smooth muscles and is protective against the adhesion of leukocytes and platelets to the blood vessel wall. However, excessive release of NO by cells in response to stimuli such as bacterial LPS, IL-1, and TNF-α can destroy infected tissue and impart a discrete cellular response. The various pro-inflammatory properties at higher levels are associated with increased vasodilatation, vascular leakiness, vascular collapse, direct cytotoxicity, and production of toxic peroxynitrite. Monocytes of elderly people showed higher NO level after stimulation meaning that the elderly had a prolonged NO production as compared to controls. Our results are supported by other similar studies. However, contradictory results have also been reported. The difference could be due to changes in microenvironment where relatively higher doses of INF-γ resulted in higher NO production from elderly monocytes when compared with young. Increased production of NO can affect various aspects of immunity. NO is a very short-aged molecule (t1/2 15 s in vivo), contributes in fine-tuning of inflammatory response. Being if among first few mediators (IL-1 being another) to be involved in inflammation, in normal inflammatory response, its level should go down by 48 h (as shown for control Group 2). The persistent higher levels as found in the study group may be due to persistent increased production and are associated with deleterious effects of inflammation such as shock/hypotension in acute state. It is a pro-inflammatory cytokine of two forms – IL-1α and IL-1 β synthesized by microbial stimulation from mononuclear cells. IL-1α functions as autocrine messenger in cytosol while IL-1 β is released into extracellular space and circulation. IL-1 is responsible for neutrophil and macrophage movement to inflammation sites; it also increases adhesion molecules' expression and acute-phase proteins' production leading to hypotension, fever, and anorexia. Our study found that the basal production of IL-1 was significantly higher in the elderly when compared to younger control. This increased level of IL-1 showed a low-grade chronic inflammatory state and may be associated with increased incidence of hypertension, congestive heart failure (CHF), and angina. Increased levels of IL-1 are also associated with Alzheimer's disease. However, 48 h following activation, the IL-1 production reduced in the elderly, indicating that the elderly monocytes failed to mount a response to LPS which is equivalent to bacterial infection in vivo. Our results were supported by Gabriel et al. This altered response could be explained by a higher basal level of IL-1 which was found to impair LPS response (LPS desensitization). The phenomenon of LPS desensitization is Known, although it has a well-controlled active response which is orchestrated to prevent excessive inflammation. However, in the elderly, it appears to inhibit even a proper inflammatory response. This in part can explain increased incidence of asymptomatic infections in the elderly. IL-6 has both pro- and anti-inflammatory properties. Although IL-6 causes fever and increased acute-phase reactant production, it does not cause shock or a capillary leakage syndrome as observed with IL-1 and TNF-α. IL-6 inhibits LPS-induced TNF-α production and increases the production of IL-1 receptor antagonist, i.e. soluble TNF-α receptor, both having anti-inflammatory properties. On the contrary, circulating TNF-α and IL-1 increases the secretion of IL-6. High levels of IL-6 have been observed in the elderly and concluded as dysregulated IL-6 production, a normal consequence of aging. We found no difference at basal as well as LPS-stimulated IL-6 production between the control and study groups. Our results are supported by Beharka et al. while studies of Fagiolo et al. contradict our results. The difference in results could be due to the selection of healthy controls. Increased IL-6 levels is associated with ageing diseases such as sarocpenia, fraility, obesity, insulin resistence, cancer, and alzheimers disase.,, Hence, it will be wise to associate the age-related diseases as a cause of increased IL-6 production, not the reverse. IL-10 is the most important anti-inflammatory cytokine of human immune response. IL-10 inhibits the production of monocytes-/macrophage-derived TNF-α, IL-1, IL-8, IL-12, and granulocyte colony-stimulating factor. IL-10 also inhibits major histocompatibility complex Class II molecule expression., However, therapeutic results of IL-10 to treat chronic inflammatory diseases including inflammatory bowel disease, rheumatoid, and psoriasis are not enthusiastic. In our study, the people in the study group had higher IL-10 production on stimulation (P > 0.05, not significant) while the control groups showed a significantly lower IL-10 production (P = 0.06 and 0.005, respectively) although the basal levels were similar, as IL-10 has anti-inflammatory properties and younger healthy controls did not show rise in IL-10 production by 48 h. Increased IL-10 production by the elderly could be a premature anti-inflammatory response, possibly due to persistent higher NO level so that severe inflammation is not required to clear the bacteria/bacterial products. As IL-10 has also been considered to be responsible for LPS desensitization, it appears that increased IL-10 level may be responsible for prolonged infections without appropriate immune response in the elderly. TNF exists in two forms, TNF-α and TNF-β. The TNF-α is more active in various inflammatory conditions and mainly produced by monocytes and macrophage. After binding to TNF-α receptor, it leads to inflammatory response resulting in enhanced leukocyte activity, increased acute phase protein synthesis, fever, anorexia, capillary leak syndrome, and increased natural killer cell function. TNF-α has been implicated in multiple age related diseases such as sarcopenia, insulin resistance, diabetes, and cancer.,, Our study showed that the basal level of TNF-α was lower in the study groups (not significant); there was a significant increase in TNF-α level after activation similar to Fagiolo et al. while results of Gabrial et al. contradict our results. Exaggerated TNF-α production results in hyperacute fatal outcome in the elderly, and disharmony in other markers may cause propensity of chronic disease.,
| Conclusions|| |
Immunosenescence and inflamm-aging could be the origin of most of the diseases of the elderly, such as infections, cancer, autoimmune disorders, and chronic inflammatory diseases. This study shows that aging is associated with alterations in various immunological parameters, both qualitative as well as quantitative, leading to a deregulated immune response. Taking all the above-mentioned facts regarding our study into consideration, we would like to propose that there is a qualitative as well as quantitative defect in elderly monocytes/macrophages' functions. Younger people go through a proper orchestration of immune response by macrophages in the following steps:
- NO production
- Increased IL-1 and TNF-α production
- Increased IL-6 and IL-10 production, the elderly immune system does not follow the same sequence.
In elderly the increased NO production, decreased IL-1 production, and increased IL-10 production at 48 h meaning thereby it is more in disharmony and there is also improper production of various inflammatory mediators. All this may finally lead to an improper, sometimes insufficient acute inflammation, prolonged inflammatory period as is seen in clinical situations following acute infections. This type of immune response may also be responsible for increased frequency of chronic diseases such as diabetes, hypertension, CHF, asymptomatic infections, and Alzheimer's disease. Identification of qualitative as well as quantitative deregulation in immune response may lead to the development of proper treatment protocol for the elderly.
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Conflicts of interest
There are no conflicts of interest.
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