Described mechanisms of lymphopenia include impaired maturation and differentiation of lymphocytes, inhibition of lymphocyte release from lymphoid tissues, enhancement of lymphocyte migration into tissues as well as death of lymphocytes with increased sensitivity to complementmediated cytolysis, activation of apoptosis and necrosis. Living in the North requires special adaptations to low temperatures resulting in a decrease in organism's reserve abilities. Cold exposure affects the thymus that is manifested as thymus hypotrophy, reducing lymphocyte abundance and increased intensity of apoptosis, which is further recorded as lymphopenia in peripheral blood [1, 2]. Cold stress leads to depletion of lymphoid tissue of mucous membranes with increasing degenerative processes thus reducing the effectiveness of defense at the "entrance gate" of infection [3, 4]. Decreased activity of cellular and humoral reactions in northern inhabitants is manifested in a higher frequency of acute and chronic infectious diseases, allergies, autoimmune processes and malignancies [5–7]. In pathological state, lymphopenia is accompanied by high levels of proinflammatory cytokines IL-6 and TNFα leading to activation of lymphocyte apoptosis and forming a destructive positive feedback loop [8]. Asymptomatic lymphopenia is often detected in people living in environmentally adverse areas and extreme climatic conditions; in the North, during periods of minimum daylight hours, the reported frequency of lymphopenia in working-age adults is up to 19.86% [9–11]. Adverse climatic effects exert stress on the body and impare the immune system. Prolonged decrease in the number of functionally active lymphocytes providing protective immune reactions significantly increases the risk of severe infectious diseases and their transition to chronic forms. In response to cold exposure, metabolic activity changes most rapidly with an increase in such biochemical parameters as concentrations of free fatty acids, C-reactive protein, glucose, etc. [12]. Factors of innate immunity are most resistant to the influence of cold exposure while for lymphocytes, glucose is a necessary substrate to increase their energy supply and active functioning. The aim of the study was to compare the characteristics of immune reactivity in response to total body cold exposure depending on background level of lymphocytes.

METHODS

Hematological and immunological parameters were measured before and immediately after total body cold exposure in two groups of volunteers (203 participants in total) depending on a background level of peripheral blood lymphocytes. The study included practically healthy individuals of working age who had no acute diseases or exacerbation of chronic diseases during the study period as well as previously and/or currently not engaged in hardening.  Persons of working age who had acute chronic diseases and their exacerbations during the study period as well as previously or currently engaged in hardening were excluded accordingly. In the trial, the participants spent 5 minutes in USHZ-25N cold chamber (Xiron-Kholod; Russia) at –25 °C in cotton clothes under constant video monitoring. The first group of participants had a background lymphopenia (n = 70, including 59 women and 11 men; lymphocyte count 1.26 (1.09–1.37) × 10⁹/L). The second group included participants having a normal lymphocyte count (n = 133, including 94 women and 39 men; with lymphocyte count 2,08 (1,81–2,45) × 10⁹/L (р^1–2 < 0,0001)). Skin temperatures at forehead and backside of palm, blood pressure and heart rate were measured before and immediately after the cold exposure. Blood samples were collected by qualified staff before and immediately after staying in cold chamber, from the ulnar vein using Vaccuette Blood Collection tubes (with EDTA for plasma and hematologic parameters; with clotting activator for getting serum). Serum and plasma were separated by centrifugation. The samples were frozen once at –20 °С. Hemograms and leukograms were determined using Automated Hematology Analyzer XS-500i (Sysmex; Japan). Ferritin (ORGENTEC Diagnostika; Germany), lactoferrin (HycultBiotech; USA), transferrin (AssayPro; USA), IL6, IL1β and TNFα (Bender MedSystems; Austria), erythropoietin (Vector Best; Russia), irisin (BioVendor; Czech Republic) levels were measured and analyzed using Thermo Scientific™ Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific; Finland). The amounts of lymphocytes undergoing apoptosis or necrosis were determined by flow cytometry on Epics XL flow cytometer analyzer (Beckman Coulter; USA) by double staining with annexin-V (AnV) and propidium iodide (PI), counting at least 5000 cells. The results were evaluated by cell staining: live cells (AnV–/PI–), apoptosis (AnV+/PI–), necrosis (AnV–/PI+). Statistical analysis was carried out using Statistica 6.0 software package (StatSoft; USA). Shapiro–Wilk test was used for testing the normality of data.  The data were presented as mean (M) ± standard deviation (SD) values. If the distribution was close to normal, t-test was used to compare the results; differences were considered significant at р < 0.05. If the distribution differed from normal, data were presented as median (Me) and 25–75% quartiles. Statistical significance of differences was assessed using the non-parametric Mann-Whitney test. The critical level of significance (р) for testing statistical hypotheses was assumed to be 0.05.

RESULTS

The individuals with lymphopenia had a decreased level of neutrophils in peripheral blood: 2.44 (1.93–2.93) × 10⁹ /L, with 38.57 ± 2.29% frequency of neutropenia. In individuals with physiologic lymphocyte levels, higher levels of neutrophil granulocytes were observed (3.02 (2.33–3.64) × 10⁹/L (p < 0.001), neutropenia was detected in 15.79 ± 1.88% of cases. Low count of neutrophil granulocytes is associated with a decrease in their phagocytic activity. Thus, the rate of active phagocytes amounted to 68.53% in the first group (lymphopenia) while in practically healthy participants is was 72.25%. The neutrophil-tolymphocyte ratio (NLR) was found to be 2.11 in group 1 and 1.49 in group 2. NLR is an important marker of conditions accompanied by systemic inflammation. Elevated NLR is known to be associated with infections, stroke, heart attack, cancer, autoimmune diseases, tissue damage and higher risk of morbidity [13–17]. In both groups, NLR was less than 3.0 which is normal. However, the higher NLR values in the group with lymphopenia evidenced imbalance of immune pathways of inflammation and can be considered as a criterion of increased systemic inflammation risks.

Assessement of apoptosis (AnV+/PI–) and lymphocyte necrosis (AnV–/PI+) showed that necrotic cells counts did not differ significantly in two groups: 0.74 % AnV–/PI+ lymphocytes in group 1, and 0.67% in group 2. The number of lymphocytes labeled for apoptosis was higher in individuals with normal lymphocyte levels in peripheral blood (5.43%), individuals with lymphopenia had 3.68% AnV+/PI– lymphocytes (p < 0.01). Thus, lymphopenia in this case is not associated with increased levels of cell death — it is rather a variant of compensatory adaptive reaction, and the impact of adverse factors leads to a shift of parameters out of normal physiological range.

In lymphopenia hemograms, we observed lower counts of erythrocytes (4.41 (4.08–4.73) and 4.68 (4.31–4.99) × 10⁶ /L, respectively, p < 0.001) and hemoglobin (127, 70 (118.00–138.00) and 137.19 (128.00–149) g/L, respectively, p < 0.0001) with no significant differences in mean hemoglobin concentrations in erythrocytes (340.33 (331.00–351.00) and 340.71 (332.00– 349.00) g/L). The frequency of detection of less than 120 g/L hemoglobin concentration was 30.75 ± 2.15% in the first group and 16.67 ± 1.46% in the second group. Less than 4 × 10⁶ erythrocytes/L values were actually four times more often detected in lymphopenia (in 20.51% and 6.06% of the participants, respectively). No significant differences in erythropoietin concentrations were observed in the two groups; it was 30.02 (13.25–35.48) mMe/mL in individuals with low lymphocyte counts and 29.68 (17.31–37.11) mMe/mL in individuals with normal lymphocyte counts.

Absorption and accumulation of iron play an important role in regulation of erythropoiesis as well as in adaptation to cold. Ferritin can serve as an indirect marker for total body iron store. Transcription of the ferritin H isoforms mRNA and accumulation of ferritin were enhanced by cold acclimation [18]. Levels of this iron-containing protein in lymphopenia occurred to be within the physiological norm with a tendency to lower concentrations as compared with individuals with normal lymphocyte counts (table). No statistically significant differences in lactoferrin levels were found for the two groups. Lymphopenia is associated with the almost 2-fold higher transferrin levels in peripheral blood. Hypoxia and low temperatures are factors enhancing the expression of the transferrin gene and, consequently, transferrin blood level. High levels of transferrin, on the one hand, increases iron supply to tissues to compensate for oxygen deficiency but on the other hand, transferrin promotes the activation of thrombin which increases the risk of hypercoagulability and as a consequence, thromboembolic and cardiovascular pathologies [19, 20].

In both groups, levels of cytokines in peripheral blood were within the physiologic norm, no significant differences were found. In case of asymptomatic lymphopenia, IL6 levels were 2.48 ± 0.41 pg/mL in group 1 and 3.74 ± 0.35 pg/mL in group 2, IL1β levels 5.01 ± 0.61 and 4.46 ± 0.67 pg/mL, and TNFα levels 6.32 ± 1.03 and 7.32 ± 0.91 pg/mL, respectively. 

After the cold exposure, an adaptive response of the cardiovascular system was recorded in both groups, with a tendency to increase blood pressure and decrease heart rate (figure).

In both groups, a significant decrease was observed in forehead skin temperatures (from 36.6 ± 0.06 to 34.05 ± 0.45 °C in group 1, p < 0.0001; from 36.4 ± 0.10 to 33.78 ± 0.32 °C, in group 2, p < 0.0001) as well as in back of the palm skin temperatures (from 33.2 ± 0.33 to 32.4 ± 0.24 °C in group 1, p < 0.05; from 33.61 ± 0.36 to 32.54 ± 0.23 °C in group 2, p < 0.05). Total body short-term cold exposure was accompanied by an increase in lymphocyte levels in individuals with lymphopenia up to 1.32 (1.13–1.48) × 10⁹ /L (p < 0.05), with no significant change in the “control” group: 2.02 (1.76–2.35) × 10⁹ /L. For neutrophil granulocytes, the opposite response was observed. In group 2 (normal lymphocyte levels) concentration of neutrophils increased by 11% up to 3.27 (2.57–4.01) × 10⁹/L (p < 0.05) while in group 1 (lymphopenia) no changes in neutrophil content were observed. In both groups there was a decrease in the level of irisin (from 4.25 (1.81–6.70) to 3.52 (1.30–5.75) μg/mL in group 1; from 2.99 (1.63–7.14) to 2.38 (1.65–5.66) μg/mL in group 2 which may be an evidence of activating mechanisms of non-shivering thermogenesis.

Cold exposure was associated with an increase in ferritin concentration up to 57.67 (41.67–65.10) ng/mL(p < 0.01) in group 1 (lymphopenia) and up to 76.46 (25.29–98.29) ng/mL (p < 0.01) in group 2 (normal lymphocyte level); lactoferrin content increased in group 2 only (up to 498.85 (124.68–485.97) (p < 0.0001); transferrin content did not change in both groups — 558.60 (421.70–940.70) mg/dL in group 1 and 423.30 (351.40–549.60) mg/dL in group 2).

DISCUSSION

Adaptive abilities depend on the levels of reactivity and resistance providing stress reaction or training reaction in response to environmental factors. Lymphopenia is known to be a negative prognostic marker in various pathological conditions, and its combination with the need to adapt to low temperatures leads to overstress of regulatory systems and failure of adaptation [21–23]. Asymptomatic lymphopenia commonly found in people living in the North is accompanied by neutropenia and lower activity of phagocytic defense.

Redistribution of leukocytes and increased transfer of neutrophil granulocytes to tissues under the influence of adverse climatic factors plays a role in the etiology of neutropenia in northerners [24, 25]. Low lymphocytes counts in peripheral blood are associated with tissue hypoxia which may be a consequence of abnormal morphofunctional state of erythrocytes caused by exposure to low temperatures and oxidative stress [26–28].

In addition, people living in the North are characterized by structural changes in erythrocyte membranes with increased membrane viscosity, and as a consequence, a decrease in the rate of gas diffusion and oxygen supply to tissues [29–31]. NLR is shown to be higher in individuals with lymphopenia as compared with individuals having normal lymphocyte levels. Combination of high NLR with insufficient oxygen supply is an adverse marker in patients with infectious inflammatory diseases [32]. In response to cold exposure, similar cardiovascular reactions have been observed in both groups regardless of the background level of lymphocytes, that are manifested in increased blood pressure and decreased heart rate. Decreased level of irisin, a protein involved in metabolism and thermoregulation is an evidence of activating thermoregulatory mechanisms [33, 34]. Irisin upregulates expression of uncoupling protein 1 (UCP1) and leads to non-shivering thermogenesis and increased heat production [35]. No changes in lactoferrin concentration have been found in individuals with background lymphopenia after the short-term cold exposure. An increase in the concentration of this protein is associated with degranulation of neutrophils and reflects the level of their activation. Thus, lymphopenia combined with neutropenia and decreased functional activity of neutrophil granulocytes significantly increases the risk of adaptive failure, and systematic exposure to cold increases the probability of chronicization of infectious diseases. In addition, a high level of transferrin persisting after cold exposure increases risk of hypercoagulability, thrombosis and cardiovascular events. In both groups, actually the same increase in ferritin levels was observed (in + 21.10% in group 1 and + 19.96% in group 2) which may be an evidence of thermal homeostasis since the induction of ferritin heavy chain expression promotes survival in cold environments by detoxifying iron forms that generate reactive oxygen species [35].

CONCLUSIONS

Asymptomatic background lymphopenia is associated with insufficient oxygen supply and higher levels of neutropenia. Regardless of the background level of lymphocytes in peripheral blood, same-type responses to short-term cold exposure are observed in cardiovascular system as well as in irisin and ferritin levels, providing an evidence of activating thermoregulation mechanisms. Cold exposure did not induced activation of nonspecific defense and did not change level and functional activity of circulating neutrophil granulocytes that can increase risks of chronicization of infectious processes in this group. The obtained data can be used in monitoring related to environmental physiology, for developing methods for assessing risks of maladaptive reactions to cold exposure and correcting immunity disorders in people living in the North.