concanavalin-a and Hypertension

Gap junctions (GJs) formed by connexins (Cxs) in T lymphocytes have been reported to have important roles in the T lymphocyte‑driven inflammatory response and hypertension‑mediated inflammation. Estrogen has a protective effect on cardiovascular diseases, including hypertension and it attenuates excessive inflammatory responses in certain autoimmune diseases. However, the mechanisms involved in regulating the pro‑inflammatory response are complex and poorly understood. The current study investigated whether β‑estradiol suppresses hypertension and pro‑inflammatory stimuli‑mediated inflammatory responses by regulating Cxs and Cx‑mediated GJs in peripheral blood lymphocytes. Male, 16‑week‑old spontaneously hypertensive rats (SHR) and Wistar‑Kyoto rats (WKY) rats were randomly divided into the following three groups: WKY rats, vehicle (saline)‑treated SHRs, and β‑estradiol (20 µg/kg/day)‑treated SHRs. β‑estradiol was administered subcutaneously for 5 weeks. Hematoxylin and eosin staining was performed to evaluate target organ injury. Flow cytometry and ELISA were used to measure the populations of T lymphocyte subtypes in the peripheral blood, and expression of Cx40/Cx43 in T cell subtypes, and pro‑inflammation cytokines levels, respectively. ELISA, a dye transfer technique, immunofluorescence and immunoblotting were used to analyze the effect of β‑estradiol on pro‑inflammatory cytokine secretion, Cx‑mediated GJs and the expression of Cxs in concanavalin A (Con A)‑stimulated peripheral blood lymphocytes isolated from WKY rat. β‑estradiol significantly decreased blood pressure and inhibited hypertension‑induced target organ injury in SHRs. Additionally, β‑estradiol treatment significantly improved the immune homeostasis of SHRs, as demonstrated by the decreased percentage of cluster of differentiation (CD)4+/CD8+ T‑cell subset ratio, reduced serum levels of pro‑inflammatory cytokines and increased the percentage of CD4+CD25+ T cells. β‑estradiol also markedly reduced the expression of Cx40/Cx43 in T lymphocytes from SHRs. In vitro, β‑estradiol significantly suppressed the production of pro‑inflammatory cytokines, reduced communication via Cx‑mediated gap junctions and decreased the expression of Cx40/Cx43 in Con A‑stimulated lymphocytes. These results indicate that β‑estradiol attenuates inflammation and end organ damage in hypertension, which may be partially mediated via downregulated expression of Cxs and reduced function of Cx‑mediated GJ.

Topics: Animals; Blood Pressure; Concanavalin A; Connexin 43; Connexins; Cytokines; Estradiol; Gap Junctions; Gene Expression; Hypertension; Inflammation; Inflammation Mediators; Kidney; Lymphocyte Activation; Lymphocytes; Male; Rats; Vascular Remodeling

Recently it has been suggested that Mg deficiency may play a key role in hypertension and several cardiovascular diseases. In order to investigate the status of Mg in genetic hypertension, the cytosolic free Mg2+ concentration ([Mg2+]i) in the lymphocytes and serum concentrations of free Mg2+ and total Mg were measured in spontaneously hypertensive rats/Izumo (SHR/Izm), stroke-prone spontaneously hypertensive rats/Izumo (SHRSP/Izm), and Wistar-Kyoto rats/Izumo (WKY/Izm). In addition, the basal cytosolic free Ca2+ concentration ([Ca2+]i) was assessed in the three strains. Systolic blood pressure was highest in SHRSP/Izm and lowest in WKY/Izm. No significant differences were found in either the serum free Mg2+ concentrations or the serum total Mg concentrations among WKY/Izm, SHR/Izm, and SHRSP/Izm. [Mg2+]i in the lymphocytes was significantly higher in SHR/Izm than in WKY/Izm (254 +/- 51 versus 201 +/- 36 mumol/liter, p < 0.05), but the [Mg2+]i in SHRSP/Izm (211 +/- 34 mumol/liter) was at the same level as in WKY/Izm. No significant correlation was found between [Mg2+]i in the lymphocytes and systolic blood pressure. Basal [Ca2+]i did not differ among the three strains. Thus, an increase in [Ca2+]i is not obligatory in all cells of genetically hypertensive rats. Mg deficiency may not exist in the intracellular or extracellular space in genetically hypertensive rats.

Topics: Animals; Calcium; Concanavalin A; Hypertension; Intracellular Fluid; Magnesium; Rats; Rats, Inbred SHR; Rats, Inbred WKY; T-Lymphocytes

We previously showed that the lymphocyte proliferation response was significantly suppressed in spontaneously hypertensive rats (SHR) and that this depressed response was due to excessive production of nitric oxide (NO) in macrophages and vascular smooth muscle cells (VSMC). Whether lymphocyte depression and activation of NO synthesis are related to age and development of hypertension remains unclear. The present study addresses such a correlation by examining the time course of development of hypertension, NO synthesis alteration and lymphocyte depression in SHR. Our results show that 1) SHR spleen cell proliferation responses are depressed at 4, 8, and 12 weeks and 1 year of age, with the lowest response occurring at 4 weeks of age; 2) this depressed response is corrected by either NO synthase inhibitor or removal of macrophages from spleen cells; 3) NO production by SHR spleen macrophages is significantly higher in all age groups; 4) upon stimulation with lipopolysaccharide or cytokines, SHR VSMC produce a significantly greater amount of NO in all age groups; 5) the increase in NO synthesis in VSMC correlates significantly with the rise in blood pressure in SHR. However, statistical correlation analysis suggests that lymphocyte depression and the alteration of NO synthesis in macrophages were not associated with either age or increased blood pressure in SHR. On the contrary, the activation of NO synthesis in VSMC can be statistically correlated with elevated blood pressure throughout the development of hypertension in SHR. Nevertheless, the results also suggest that a general alteration in the NO synthesis system may exist in SHR.

Topics: Aging; Animals; Arginine; Blood Pressure; Cell Division; Cells, Cultured; Concanavalin A; Enzyme Inhibitors; Hypertension; Lymphocytes; Macrophages; Male; Muscle, Smooth, Vascular; Nitric Oxide; omega-N-Methylarginine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Species Specificity; Spleen

Aspects of T and B cell function were studied in women with pregnancy-induced hypertension (PIH) and normotensive pregnant women by determining the proliferation of peripheral blood mononuclear cells (PBMC) with or without stimulation by mitogens (PHA, ConA and PWM) and by determining IgG and IgM levels in the culture supernatant. The results showed that the proliferation of PBMC without mitogens was significantly increased in PIH women without proteinuria compared with normotensive pregnant women. In the presence of PHA, [3H]thymidine uptake in PBMC was statistically higher in PIH women both with and without proteinuria than that in normotensive pregnant women. ConA and PWM mitogen activities were not significantly different between PIH women and normotensive pregnant women. Compared with normotensive pregnant women, IgG production was significantly increased in PIH women with proteinuria but not in those without proteinuria. IgM production was not changed in PIH women. We concluded that immunologic responses in PIH women were increased rather than decreased. This increased immunologic activity is in accordance with some important changes seen in PIH, such as an increase in intracellular calcium, the presence of blood-borne mitogenic factor and a decrease in prostaglandin E series. These findings also support the hypothesis that PIH might result from the imbalance between fetal antigenic load and maternal production of immunologic blockade.

Topics: Adult; B-Lymphocytes; Concanavalin A; Female; Humans; Hypertension; Immunoglobulin G; Immunoglobulin M; Lymphocyte Activation; Phytohemagglutinins; Pokeweed Mitogens; Pregnancy; Pregnancy Complications; T-Lymphocytes

The immune system of the spontaneously hypertensive rat is dysfunctional compared with that of normotensive control strains. Previous studies from our laboratory have shown that immunodepression in the spontaneously hypertensive rat was mediated by macrophages. The current study examines the mechanism for the depressed proliferative responses to concanavalin A typically observed by splenic mononuclear cells of spontaneously hypertensive rats. We tested various inhibitors of known macrophage products responsible for suppressing lymphoid function. The nitric oxide synthetase inhibitor NG-monomethyl L-arginine produced dose-dependent derepression of the proliferative responses of splenic mononuclear cells to concanavalin A. In contrast, indomethacin and catalase exhibited only weak derepression of the proliferative responses. Subsequent analysis showed that splenic mononuclear cells from spontaneously hypertensive rats generated greater nitric oxide levels than cells from Wistar-Kyoto rats, and nitric oxide levels were reduced when the inhibitor was added to splenic mononuclear cell cultures from spontaneously hypertensive rats. We further demonstrated that L-arginine is required for the development of the depressed mitogen-induced proliferative responses in these cells. Addition of L-arginine in excess of 10 microM to cultures diminished cell proliferation and increased nitric oxide. Polyclonal antibodies to murine interferon gamma reduced nitric oxide accumulation by approximately 50%, suggesting that interferon gamma is partially responsible for enhancing nitric oxide production in mitogen-stimulated splenic mononuclear cell cultures from spontaneously hypertensive rats. Thus, this study provides evidence that the immune depression observed in the spontaneously hypertensive rat is nitric oxide dependent.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cell Division; Concanavalin A; Hypertension; Immune System Diseases; Interferon-gamma; Leukocytes, Mononuclear; Male; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Spleen

Changes of membrane potential of lymphocytes of peripheral blood under the influence of the activation agents: phytohemagglutinin, concanavalin A and Ca(2+)-ionophore A 23187 in 25 patients (men) with hypertensive disease and 8 healthy persons (men) aged 25 to 35 years have been studied. The results obtained testify to the existence of functional disturbances of lymphocytes' membrane and confirmed the wide-spread of the membrane defect in patients with hypertensive disease.

Topics: Adult; Calcimycin; Concanavalin A; Humans; Hypertension; Lymphocyte Activation; Lymphocytes; Male; Membrane Potentials; Phytohemagglutinins

It has been shown that spontaneously hypertensive rats (SHR) exhibit some abnormalities in their immune system. These include a reduced delayed hypersensitivity response, a reduction in the number of rosette-forming cells and a decreased lymphocyte blastogenic response. In this study, we further investigated the blastogenic responses of splenocytes, thymocytes, and T-enriched lymphocytes from SHR. In SHR splenocytes, the blastogenic responses to concanavalin A (Con A), phytohemagglutinin (PHA), interleukin-2 (IL-2), and phorbol 12,13-dibutyrate (PDB) plus ionomycin were significantly reduced compared with those from Wistar-Kyoto rats (WKY). In SHR thymocytes and T-enriched lymphocytes, the blastogenic responses to these activators were the same as in WKY rats. The IL-2 production by SHR splenocytes was similar to that of WKY. To elucidate the possible mechanism responsible for the blastogenic defects in SHR splenocytes, the involvement of the nitric oxide (NO) synthetic pathway was studied. The inhibition of NO synthesis by NG-monomethyl-L-arginine (L-NMMA) corrected the defect in SHR splenocytes. L-NMMA had no effect on the splenocytes, thymocytes, or macrophage-depleted splenocytes from WKY or on thymocytes or macrophage-depleted splenocytes from SHR. The removal of macrophages from SHR splenocytes also corrected the blastogenic defect in these cells. Furthermore, the NO synthesis in Con A stimulated SHR splenocyte culture medium was statistically significantly higher than that in WKY. These results suggested that overproduction of nitric oxide by SHR macrophages may be responsible for the SHR splenocyte blastogenic defect.

Topics: Animals; Arginine; Concanavalin A; Hypertension; Interleukin-2; Lymphocyte Activation; Lymphocytes; Male; Nitric Oxide; omega-N-Methylarginine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Spleen; Thymus Gland

Suppressor T cell function in the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats was analyzed using syngeneic mixed lymphocyte reaction (SMLR) and concanavalin A (Con A) activation. A depressed SMLR was found in adult SHR but not in adult WKY. IL-2 synthesized by SHR was 40-fold lower than that of WKY, and the suppressor T cells generated in the SMLR were incapable of suppressing IgG synthesis. Precursors of cells that can be activated by Con A to become functional suppressor cells are reduced in adult SHR. Supernatant fluids derived from Con A-activated spleen cells from adult SHR failed to significantly inhibit IgG synthesis by cultures of syngeneic spleen cells compared to supernatant fluids from young SHR or WKY Con A-activated spleen cells. However, spleen cells from both adult SHR and WKY proliferated strongly and released equivalent amounts of IL-2 in response to Con A. Addition of exogenous IL-2 to the SMLR cultures in vitro restored the ability of SHR T cells to respond in the SMLR, with generation of cells capable of suppressing IgG synthesis. Administration of SHR with IL-2 in vivo also restored the suppressor T cell function in the SMLR. These results suggest a defective suppressor T cell activation and loss of suppressor T cell activity as the SHR age.

Topics: Animals; Cells, Cultured; Concanavalin A; Hypertension; Immunoglobulin G; Indomethacin; Interleukin-2; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Male; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Spleen; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory

The plasma and adrenal renin-angiotensin system in stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats were examined in animals at 5, 11, 18, and 25 weeks of age. Plasma active renin was significantly increased in 18- and 25-week-old SHRSP with impaired renal function, whereas there was no difference in the plasma prorenin level or renal renin content between the two strains at all ages examined. Thus, the rate of activation of prorenin seems to be enhanced in the kidney of SHRSP with malignant hypertension. Adrenal renin contents were severalfold higher in SHRSP than WKY rats at all ages. However, adrenal angiotensin peptides were not increased in SHRSP aged 5 and 11 weeks. In 18-week-old SHRSP, adrenal angiotensin II (Ang II) and III (Ang III) levels were fourfold and 1.8-fold higher, respectively, than in WKY rats, accompanied by 1.5-fold higher plasma aldosterone. Increased adrenal angiotensin and plasma aldosterone were also found in 25-week-old SHRSP. Zonal distribution studies indicated that the elevated Ang II and III in SHRSP were derived mainly from the capsular tissue (the zona glomerulosa). To examine the contribution of circulating angiotensin to the adrenal angiotensin content, effects of bilateral nephrectomy on adrenal angiotensin and renin were examined in 18-week-old rats. At 24 hours after nephrectomy, plasma angiotensin, prorenin, and active renin were decreased to almost negligible concentrations. Conversely, in both adrenal capsular and decapsular tissues of SHRSP and WKY rats, neither angiotensin nor renin was significantly decreased after nephrectomy. These results suggest that the increase in adrenal capsular Ang II contents in SHRSP may be partly due to an enhanced local production of Ang II.

Topics: Adrenal Glands; Aldosterone; Angiotensin II; Animals; Cerebrovascular Disorders; Concanavalin A; Enzyme Precursors; Hypertension; Immune Sera; Male; Nephrectomy; Radioimmunoassay; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Renin; Renin-Angiotensin System

Immune dysfunction has been reported in spontaneously hypertensive rats (SHR), particularly in mature animals with established hypertension. The current study examined the time course of development of immune dysfunction and defined its cellular basis in male SHR and control normotensive Wistar-Kyoto rats (WKY). Mitogen-induced proliferative responses in lymphoid cells obtained from induced proliferative responses in lymphoid cells obtained from SHR thymus and spleen before (age 4 wk) and during the development of (ages 8 and 12 wk) hypertension and in age-matched normotensive WKY were monitored. A 50% reduction in concanavalin A (Con A)-induced proliferative responses was seen in SHR thymocytes compared with those of WKY at 12 wk only, suggesting differences in immature T-cell populations. Con A-induced T-cell proliferative responses in splenocytes also differed between strains: greatest (as much as 8-fold) decreases were found in 12-wk-old SHR. Similar findings were obtained in splenocytes stimulated with lipopolysaccharide (LPS), indicating differences in B-cell function. Mononuclear cells depleted of their adherent cell population were prepared from SHR and WKY at 12+ wk of age and assayed for their proliferative responses to LPS and Con A. The remaining nonadherent mononuclear cells of SHR had proliferative responses equal to or greater than those of WKY. Further, when SHR splenic mononuclear cells were allowed to adhere to plastic, and the adherent fraction was co-cultured with either SHR G-10 nonadherent or unfractionated SHR splenic mononuclear cells, proliferative responses were suppressed by as much as 88%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; B-Lymphocytes; Cell Adhesion; Concanavalin A; Hypertension; Immune System Diseases; Lipopolysaccharides; Lymphatic System; Lymphocyte Activation; Lymphoid Tissue; Male; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Spleen; T-Lymphocytes

T cell function tests were performed in 6 patients with uncomplicated essential hypertension (EH) before and after treatment with captopril, an angiotensin-converting enzyme inhibitor. Total T and T cell subsets were within normal range and were not affected by the drug. The response of PBL to lectin stimulation was significantly impaired. While the stimulation index (SI) was almost normal when washed PBL were used (32.2 +/- 6.90 with PHA and 20.17 +/- 4.1 with Con A) or after their incubation with serum taken from normal subjects (40.42 +/- 10.9 and 15.53 +/- 3.4, respectively), autologous serum significantly reduced the SI (15.04 +/- 5.9 with PHA, p less than 0.05 and 6.68 +/- 1.45 with Con A, p less than 0.005). While captopril seemed to suppress the SI of washed PBL after 1 week of treatment, it enhanced the SI from 32.21 +/- 6.91 to 55.32 +/- 10.76 for PHA and from 20.17 +/- 4.13 to 30.63 +/- 5.41 for Con A (p less than 0.001). This effect was more obvious when the stimulations were performed with normal serum (from 40.42 +/- 10.9 to 96.47 +/- 17.51 for PTH and from 15.53 +/- 3.43 to 40.02 +/- 8.0 for Con A, (p less than 0.001). These results confirm previous reports indicating that the cellular immune response is impaired in EH. It seems that an inhibitory factor found in the serum of these patients is responsible for this impairment. Our findings may suggest that this factor may be angiotensin II.

Topics: Aged; Captopril; Concanavalin A; Female; Humans; Hypertension; In Vitro Techniques; Lymphocyte Activation; Male; Middle Aged; Phytohemagglutinins; Renin-Angiotensin System; T-Lymphocytes

We have reported that the basal and stimulated cytosolic free calcium concentrations [( Ca2+]i) are elevated in platelets isolated from 12-14-week-old spontaneously hypertensive rats (SHR) as compared with normotensive Wistar-Kyoto (WKY) rats. To determine whether altered cell calcium metabolism precedes the development of overt hypertension, we measured [Ca2+]i under resting and stimulated conditions in blood platelets and thymic lymphocytes isolated from 4-week-old prehypertensive SHR and WKY rats. Blood pressure was similar in both groups (SHR 95 +/- 8 versus WKY rats 92 +/- 7 mm Hg). Basal [Ca2+]i in platelets was higher in SHR than WKY rats (63.4 +/- 3.9 versus 54.8 +/- 3.1 nM, p less than 0.003). Also the [Ca2+]i response to thrombin was greater in SHR than WKY rats in both the presence and absence of extracellular calcium. For lymphocytes, although no difference was detected in basal [Ca2+]i, the concanavalin A-induced peak [Ca2+]i was higher for SHR than WKY rats in both calcium-containing and calcium-free media. These results suggest that agonist-stimulated calcium influx and calcium discharge from intracellular stores are enhanced in both platelets and lymphocytes of 4-week-old SHR. We conclude that abnormalities in calcium metabolism in two different cell types precede the development of overt hypertension in the SHR.

Topics: Animals; Blood Platelets; Calcium; Concanavalin A; Hypertension; Lymphocytes; Male; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Thrombin

Several studies in human and experimental models indicate the existence of a partial relationship between essential hypertension (EH) and the immune system. In this study, cellular immune functions were investigated in 13 patients with untreated and uncomplicated essential hypertension (EHP) and in 10 of their offspring (EHO) and compared to 13 age- and sex-matched normotensive controls (NC) and 10 of their offspring (NCO). The total number of T cells and T cell subsets were similar in all groups examined. In the EHP, basal lymphocyte transformation without lectins was significantly lower (1,126 +/- 261 cpm of [3H]-thymidine uptake) than in the NC (3,223 +/- 736, p less than 0.01); the response to both phytohemagglutinin (PHA) and concanavalin A (ConA) revealed reduced [3H]-thymidine uptake as compared with NC (21,890 +/- 5,432 compared to 64,574 +/- 9,723 for PHA and 10,488 +/- 2,621 compared to 37,334 +/- 8,148 for ConA, respectively, p less than 0.01). However, the ability to proliferate as a response to lectins was normal. This was leading to a normal stimulation index in both groups. In the EHO, non-significant decrease in basal transformation and reduced uptake with PHA (49,537 +/- 7,478) versus NCO (69,911 +/- 7,254) and NC (64,574 +/- 9,723) were found. These findings suggest that the proliferative response of T lymphocytes is partially suppressed in EH.

Topics: Adult; Aged; Concanavalin A; Female; Humans; Hypertension; In Vitro Techniques; Lymphocyte Activation; Male; Middle Aged; Phytohemagglutinins; T-Lymphocyte Subsets; T-Lymphocytes

Lymphocyte reactivity assessed by a fluorescent lipophilic probe test of responsiveness to concanavalin A (con A) was shown to differ from normal in early pregnancy. The difference was most marked in multiparas. Abnormal reactivity was detected in the earliest pregnancy examined (5 weeks' gestation) and up to about the 20th week; after 20 weeks, reactivity was normal in all of the multiparas and most of the nulliparas studied. However, in pregnancy induced hypertension (PIH), a disorder of late pregnancy, the same responsiveness as in early pregnancy was found. When unstimulated lymphocytes were examined, abnormal reactivity associated with increased fluorescence was observed in early pregnancy and in PIH, compared with normal late pregnancy, reflecting alteration in lymphocyte membrane phospholipids. It is postulated that pregnancy is associated with sequential change in immunity, disturbance of which may result in immunologically-determined obstetric morbidity.

Topics: Adult; Concanavalin A; Female; Fluorescence; Fluorescent Dyes; Humans; Hypertension; Lymphocyte Activation; Parity; Pregnancy; Pregnancy Complications, Cardiovascular