For over 50 years, it has been recognized that low-grade systemic inflammation contributes to hypertension. This can be seen in the infiltration of T lymphocytes and the elevation in the secretion of T cell-derived pro-inflammatory cytokines in the serum and target organs in different experimental hypertension models . Alterations in the expression and function of Cxs are a well-documented event of the inflammatory response and are known to contribute to changes in the proliferation and activation of T lymphocytes and in inflammatory cytokine production [24, 25]. However, these studies have not investigated the link between the expressional or functional changes in Cxs of T cell subtypes and abnormal alterations of T cell subtypes or in the increase in pro-inflammatory cytokine production in hypertension. To better understand the mechanisms of Cxs regulating hypertension-mediated inflammation, we investigated the possible regulatory effect of Cxs on alterations in the percentages of T-lymphocyte subsets, T-lymphocyte proliferation and pro-inflammatory cytokine synthesis in the peripheral blood of hypertensive rats.
Hypertensive stimuli like Ang II, high salt and excessive catecholamines lead to the formation of effector T cells, resulting in the development of prehypertension . There is evidence to suggest that factors like Ang II promote elevation of blood pressure, increase the expression of pro-inflammatory cytokines, and induce both proliferation of splenic lymphocytes and cytokine production through receptors on immune cells [3, 35]. Ang II and DOCA-salt also significantly increase vascular and renal infiltration of CD4+ and CD8+ T cells in male animals . Initial elevations in blood pressure during prehypertension may in turn lead to T-cell activation . Activated T cells and T cell-driven cytokines that cause vasoconstriction and vascular remodeling ultimate contribute to the development of hypertension . On the other hand, chronic inflammation is now recognized as a contributing factor to many age-associated diseases, including metabolic disorders, arthritis, neurodegeneration and cardiovascular disease . Indeed, several studies from hypertensive rat and mouse models showed that both CD4+ and CD8+ T cells are involved in the pathogenesis of hypertension, and CD4+ cells are the main adaptive immune players in hypertension [4, 5]. 24-week old male SHRs also showed increased helper (CD4) T cell infiltration and a high CD4+/CD8+ ratio . In our study, we compared the different lymphocyte subsets, including T-helper cells (CD3+CD4+), cytotoxic T-cells (CD3+CD8+), and Treg cells (CD4+CD25+), in the peripheral blood of spontaneously hypertensive rats (SHRs) and WKY rats. Our results showed significantly higher tail blood pressure in SHRs compared to WKY rats. We also found an increase in the accumulation of CD4+ T cells and in the CD4+/CD8+ ratio occurred in the peripheral blood of SHRs. This was evident in the increased secretion of IL-2, IL-4 and IL-6, which are the secretory products of activated T lymphocytes. Taken together with the results of the previous studies described above, these findings suggest that CD4+ T-cell irregularities contribute to the development of hypertension, with a reciprocal relationship to blood pressure elevation and cytokine production. Furthermore, hypertensive patients, Ang II-infused male Rag-1−/− mice and male SHRs exhibit significantly greater numbers of cytotoxic CD3+CD8+ T cells [5, 38]. CD8-deficient mice have a blunted hypertensive response, and adoptive transfer of CD8 into Rag1-deficient mice recovers a normal blood pressure increase during Ang II administration . However, our results and other data from our lab showed that the percentages of CD8+ T cells in the peripheral blood of SHRs and essential hypertensive patients  were reduced, which may be caused by enhanced infiltration of CD8+ T cells into other tissues. Thus, we can speculate here that the decrease in the number of activated CD8+ T cells represents general immunological dysregulation in hypertensive rats, although the cause is not entirely clear from this study. In contrast to pro-inflammatory T cells, CD4+CD25+ Treg cells with immunosuppressive capabilities are considered a blocking modulator that can ameliorate blood pressure elevation in response to Ang II or aldosterone [12, 14, 16], whereas DOCA-salt and Ang II stimuli caused a reduction in Tregs in animals [12, 39]. Thus, an imbalance between effector T lymphocytes and Tregs also represents a crucial mechanism in hypertension-mediated inflammation. Data from our laboratory has also shown that spleen of SHRs presented a significantly decreased percentage of CD4+CD25+ (Treg) T cells . We have here provided evidence that CD4+CD25+ T cells are markedly diminished in the peripheral blood of SHRs, suggesting that lower Tregs prevail in hypertension-mediated inflammation. This demonstrates that imbalance in Treg function or number improves hypertension-mediated inflammation and is an important factor in the development of hypertension.
Several pro-inflammatory cytokines (IL-2, IL-4 and IL-6) secreted by T cells were shown to be elevated in the serum of many hypertensive models and hypertensive patients, contributing to the inflammation of blood vessels [15, 16]. In this study, compared with WKY rats, SHRs had higher serum levels of IL-2, IL-4 and IL-6. IL-6 is fundamental for the development of stress-induced hypertension . Increased IL-6 levels suppress CD4+ naïve T-cell differentiation into Tregs . These findings together with our results strongly support the essential roles of IL-2, IL-4 and IL-6 in the development of hypertension.
Although considerable research, including our work, has shown that the disorder of lymphocyte subtypes plays an important role in hypertension, the precise mechanisms underlying this role remain unclear. Comprehending how T-lymphocyte subsets become imbalanced and participate in hypertension-mediated inflammation is crucial. Growing evidence indicates that Cxs-based channels play an indispensable role in modulating the clonal expansion of CD4+ T cells and the production of cytokines [28, 43]. Cx40 and Cx43 are the main Cxs in almost all immune cells, with the predominant expression of Cx43 in circulating lymphocytes  and Cx43 acts in a pro-inflammatory way [44, 45]. Recently, it was reported that activation of CD4+ T cells is associated with an upregulation of Cx43 expression . During T-cell activation, the expression of GJCs and HCs mainly constituted by Cx43 contributes to clonal expansion of T cells . Moreover, expression or accumulation of Cxs (Cx40 and Cx43) in the plasma membranes and/or cytoplasm of T lymphocytes are actively regulated by pro-inflammatory molecules such as LPS, mitogen, anti-CD3/anti-CD28 beads and numerous cytokines (TNF-α Plus IFN-γ) [25, 29, 30]. However, it is uncertain whether hypertension-mediated inflammation induces upregulation of Cxs expression in T-lymphocyte subsets, and whether Cxs are also implicated in hypertensive inflammation-induced alterations in the production of pro-inflammatory cytokines. Excitingly, our results clearly showed that surface and/or cytoplasmic expression of Cx40 and Cx43 in peripheral blood lymphocytes and CD4+/CD8+ T cells were significantly increased in SHRs. In addition, we also found a strong correlation between the serum levels of pro-inflammatory cytokines (IL-2 and IL-6) and the expression levels of Cx40 or Cx43 in SHRs. Although T, B and NK cells from secondary lymphoid organs have been shown to express Cx40 at a low level , the contribution of Cx40 to the activation and proliferation of lymphocytes is still unknown. It has been proposed that Cx40-formed hemi-channels facilitate ATP-mediated propagation of calcium ions, but this is speculative [32, 46]. Thus, the role of Cx40 in T-lymphocytes remains to be further investigated. Above all, these results provide an explanation for the importance of pro-inflammatory cytokines in the maintenance of Cxs expression, and the association between hypertension-mediated inflammation and the upregulation of Cxs expression in peripheral blood lymphocytes. Most notably, it is believed that Cxs can be upregulated when immune cells become exposed to inflammatory factors .
Cx43-based channels have been reported to be directly implicated in intercellular communication between human peripheral blood lymphocytes or PBMCs [30, 31]. The functionality of GJIC through Cx43-based channels is regulated by inflammatory stimulators (LPS and PHA) , and Cx43 expression can be induced by cytokines in monocytes and DCs [29, 47], whereas blocking of Cx43-based channels remarkably reduced cytokine secretion (IFN-γ, IL-2 and IL-10) by T cells and thereby suppressed the inflammatory response [22, 25, 31]. This evidence clearly demonstrates that the regulative relationship between Cx43-mediated cellular communication and the production of pro-inflammatory cytokines is reciprocal, which may be involved in the pathological mechanisms of hypertensive inflammation. Our previous studies have also demonstrated that pro-inflammatory cytokines (IL-2) or essential hypertension promote cellular communication in peripheral blood lymphocytes and the production of IFN-γ and TNF-α . In agreement with our previous observations, our new results also show a promoting effect of hypertension or the T-cell mitogen (Con A) on calcein dye transfer between peripheral blood lymphocytes, and enhanced mRNA expression levels of IL-2 and IL-6 in Con A-stimulated lymphocytes from SHRs. An important implication of our data is that the enhanced GJIC in lymphocytes from SHRs may be involved in the hypertensive inflammatory response. In the presence of a specific inhibitor of Cx43, we observed diminished dye coupling from calcein donor cells to receptor cells in SHRs, and reduced IL-2 and IL-6 expressions in Con A-stimulated peripheral blood lymphocytes of SHRs. This is similar to our previous findings in the lymphocytes of both essential hypertensive patients and normotensive healthy subjects . These results indicate that inhibition of Cx43-based channels may result in disrupted GJIC between peripheral blood lymphocytes and in a reduction in pro-inflammatory cytokines. This further supports a direct correlation between Cx43-mediated GJIC in lymphocytes and hypertension-mediated inflammation or the production of cytokines. The inhibition was not complete, which may be due to the high connexin membrane turnover under inflammatory stimulus by Con A. Another reason for the lack of complete inhibition of GJIC is that Gap27 may not completely inhibit heterotypic junctions constructed of Cx40/Cx43 
Although Gap27 inhibits Cx43-mediated intercellular communication, it also suppresses Cx37-based GJCs, pannexin channel currents  and Cx43-based HCs [49, 50]. However, the expression of Cx37 in the immune system has appeared only in lymphatic vessels, resting monocytes and cell lines derived from macrophages (macrophage foam cells) [20, 51]. There is also compelling evidence that Cx37 is not found in peripheral blood and tonsil lymphocytes [30, 52]. It is almost impossible for there to be any Cx37-mediated GJIC between peripheral blood lymphocytes. Therefore, Gap27 acts mainly on Cx43-mediated channels or hemi-channels in our study. The inhibitory kinetics of GJCs and HCs by Gap27 are different, with inhibition of HCs occurring more rapidly (minutes) than inhibition of GJCs (tens of minutes to hours) . Based on this observation, the incubation time of Gap27 is long enough to completely block the Cx43-based GJC-mediated inflammatory response in our study, but the involvement of Cxs- and Panx1-based HCs cannot be excluded. Thus, our results above using Gap27 incubation are very likely related to a combined effect on the inhibition of Cx43-mediated channels and hemi-channels. In contrast to GJCs, HCs are believed to specifically open up in pathological conditions and affect pro-inflammatory cytokine production and proliferation of T cells through ATP release [20, 23]. However, HCs involvement in hypertension-mediated inflammation remains to be investigated. Further studies using the HC-specific mimetic peptide Gap19 and Peptide5  in combination with lentivirus-mediated RNAi knockdown of Cx43 may discern the contribution of HCs in immune cells to hypertension-mediated inflammation.