The immune response is determined by the speed of the T cell reaction to antigens assured by a state of readiness for proliferation and cytokine secretion. Proliferation, apoptosis and motion of many cell types are controlled by cytoplasmic proteases - μ- and m-calpain - and their inhibitor calpastatin, together forming the “calpain-calpastatin system” (CCS), assumed to modify their targets only upon activation-dependent cytoplasmic Ca2+ increase. Contrastingly to this notion, using quantitative real time PCR and semiquantitative flow cytometry respectively, we show here that the CCS genes are constitutively expressed, and that both calpains are constitutively active in resting, circulating human CD4+ and CD8+ lymphocytes. Furthermore, we demonstrate that calpain inhibition in the resting T cells prevents them from proliferation in vitro and greatly reduces secretion of multiple cytokines. The mechanistic reason for these effects of calpain inhibition on T cell functions might be the demonstrated significant reduction of the expression of active (phosphorylated) upstream signalling molecules, including the phospholipase C gamma, p56Lck and NFκB, in the inhibitor-treated cells. Thus, we propose that the constitutive, self-regulatory calpain-calpastatin system activity in resting human T cells is a necessary, controlling element of their readiness for complex and effective response to antigenic challenge.
IntroductionCurrently used clinical scale and laboratory markers to monitor patients with early rheumatoid arthritis (RA) seem to be not sufficient. It has been demonstrated that disease- related cytokines may be elevated very early in RA development and cytokines are considered as the biomarkers potentially useful for RA monitoring.Material and methodsThe group of patients with undifferentiated arthritis (UA) developing RA (UA→RA) was identified from a total of 121 people with arthralgia. UA→RA (n = 16) and healthy control (n = 16) subjects underwent clinical and laboratory evaluation, including acute phase reactants (APRs) and autoantibodies. Cytokines IFN-γ, IL-10, TNF, IL-17A, IL-6, IL-1b, IL-2 in sera were assayed using flow cytometric bead array test.Results34.5% of patients with UA developed RA. DAS28 reduced as early as 3 months after initiation of treatment. No DAS28 difference between groups of autoantibody (RF, anti-CCP, ANA-HEp-2) -positive and -negative patients was observed, however, comparing groups of anti-CCP and RF-double negative and -double positive patients, the trend of sooner clinical improvement was visible in the second abovementioned group. After the treatment introduction, the ESR level reduced significantly, while CRP level reduction was not significant. Serum cytokine levels of IL-10, IL-6 and IL-17A reduced after 6 months since introduction of treatment. The positive correlations between ESR, CRP and specific cytokine levels were observed.ConclusionsThe autoantibody and APR profile is poorly connected with the RA course. The serum cytokine profile change in the course of RA and may be potentially used for optimization of RA monitoring.
Childhood acute lymphoblastic leukemia (ALL) blasts are characterized by inhibited apoptosis promoting fast disease progress. It is known that in chronic lymphocytic and acute myeloid leukemias the reduced apoptosis is strongly related with the activity of calpain-calpastatin system (CCS) composed of cytoplasmic proteases—calpains—performing the modulatory proteolysis of key proteins involved in cell proliferation and apoptosis, and of their endogenous inhibitor—calpastatin. Here, the CCS protein abundance and activity was for the first time studied in childhood ALL blasts and in control bone marrow CD19+ B cells by semi-quantitative flow cytometry and western blotting of calpastatin fragments resulting from endogenous calpain activity. Significantly higher μ-calpain (CAPN1) gene transcription, protein amounts and activity (but not those of m-calpain), with calpastatin amount and transcription of its gene (CAST) greatly varying were observed in CD19+ ALL blasts compared to control cells. Significant inverse relation between the amount/activity of calpain and spontaneous apoptosis was noted. Patients older than 10 years (considered at higher risk) displayed increased amounts and activities of blast calpain. Finally, treatment of blasts with the tripeptide calpain inhibitors II and IV significantly and in dose-dependent fashion increased the percentage of blasts entering apoptosis. Together, these findings make the CCS a potential new predictive tool and therapeutic target in childhood ALL.
Introduction.Much of what we know about the functioning of human T lymphocytes is based on the experiments carried out at atmospheric oxygen (O 2 ) concentrations, which are significantly higher than those maintained in blood. Interestingly, the gender differences in the activity of T cells and their susceptibility to apoptosis under different O 2 conditions have not yet been described. The aim of the study was to compare two main markers of lymphocyte function: proliferation capacity and ability to produce cytokines as well as their susceptibility to apoptosis under two different O 2 concentrations, between men and women. Material and methods. 25 healthy volunteers, both males (13) and females (12) were recruited to the study (mean age 25.48 ± 5.51). By using cytometry proliferation parameters of human CD4 + CD28+ cells or CD8 + CD28 + cells in response to polyclonal stimulation of the TCR/CD3 complex at atmospheric (21%) and physiological (10%) O 2 concentrations using our modified dividing cell tracking technique (DCT) were analyzed as well as the percentages of apoptotic cells. We also determined the levels of IFN-g, IL-2, IL-10 and IL-17A using Cytometric Bead Array Flex system in cell culture supernatants. Results. CD4+ CD28 + and CD8 + CD28 + cells from the whole study group were characterized by shorter time required to enter the first (G1) phase of the first cell cycle at 21% compared to 10% O 2 . Both T cell populations performed significantly more divisions at 21% O 2 . The percentages of dividing cells were also significantly higher at atmospheric O 2 . Interestingly, data analysis by gender showed that male lymphocytes had similar proliferative parameters at both O 2 concentrations while female lymphocytes proliferate more efficiently at 21% oxygen. Compared to males, the female CD4 + cells showed increased susceptibility to apoptosis at both O 2 concentrations. No differences in the levels of cytokines regardless of gender and oxygen conditions were found. Conclusions. We showed that in vitro female T cells (both CD4 + and CD8 + cells) are more sensitive than male lymphocytes to low O 2 concentration as demonstrated by the decrease in their proliferation dynamics. The effect does not depend on increased apoptosis of female T cells under low O 2 because percentage of apoptotic cells was similar at both O
We have shown before that the expression of ZNF334 gene, coding for a newly described zinc finger protein of as yet unknown function, is extremely reduced in CD4 + lymphocytes of rheumatoid arthritis (RA) patients regardless of their age, and thus can be considered a new molecular marker of the disease. Based on the promoter sequence of the gene we speculated that it might be regulated by TNFα. Here we have tested that hypothesis, studying the in vitro influence of TNFα on the ZNF334 gene expression and protein levels in resting and stimulated CD4 + cells of healthy volunteers. We have confirmed that treatment with TNFα modifies the levels of ZNF334 expression in the CD4 + cells ex vivo; however, the effect varied for different individuals and reduction of expression was seen only for those cell samples that initially exhibited high transcriptional activity of the gene, while for those exhibiting initially very low expression, some increase in the transcriptional activity was observed. Incubation with TNFα significantly reduced the amounts of two isoforms of ZNF334 protein (initially high in all subjects) in parallel to the reduced transcription. Finally, the expression of ZNF334 in CD4 + lymphocytes isolated after various periods of anti-CD3 stimulation generally increased with longer culture times, and the effect of TNFα treatment was negligible. Concluding, our results obtained in vitro for helper lymphocytes of healthy individuals seem to mimic the regulatory effect of TNFα on the expression of ZNF334 in the cells of RA patients.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.