scholarly journals AB0032 DIFFERENTIAL IMPACT OF BIOLOGICS AND GLUCOCORTICOIDS ON TNF SECRETION AND CD14+CD16+ MONOCYTES PERCENTAGE IN CULTURE DERIVED FROM SYNOVIAL FLUIDS OF PATIENTS WITH PSORIATIC ARTHRITIS-IN VITRO RESEARCH

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1049.2-1049
Author(s):  
S. Gertel ◽  
A. Polachek ◽  
V. Furer ◽  
D. Levartovsky ◽  
R. Tzemach ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Psoriatic Arthritis ◽  
Tumor Necrosis Factor ◽  
Synovial Fluid ◽  
Tumor Necrosis ◽  
Mononuclear Cells ◽  
Tnf Inhibitors ◽  
Necrosis Factor ◽  
Tnf Secretion

Background:Inflammatory joint diseases, such as psoriatic arthritis (PsA), are frequently treated by biologics. Assessment of treatment efficacy is based upon change in clinical activity scores and in tender and swollen joint counts. Although the response to these agents may be attributed to their central anti-inflammatory effects, synovial response may be operating in parallel. The potential beneficial role of intra-articular injection of tumor necrosis factor (TNF) blockers compared to glucocorticoids (GCs) in reducing synovitis was shown by means of clinical and instrumental validated measures (1). The inflamed synovial fluid is rich in mononuclear cells, however, the different mode of action of the in vitro response of these cells to drugs may contribute to the understanding of cellular response to therapeutic agents in central as opposed to peripheral compartments.Objectives:To evaluate the effect of biologics used in the management of PsA on synovial fluid mononuclear cells (SFMCs) in vitro, and to compare their modes of action to GCs that are used to locally alleviate synovial inflammation.Methods:SFMCs were obtained from PsA patients (n=11) during therapeutic knee arthrocentesis. The cells were cultured in vitro for 7 days in the presence of biologics (adalimumab, infliximab, secukinumab and ustekinumab, 10ug/ml) and GCs (betamethasone and methylprednisolone, 1 ug/ml and 10ug/ml) or medium as control. Levels of the secreted TNF were measured by ELISA. Changes in %CD14+CD16+ monocytes were analyzed by flow cytometry.Results:Both TNF inhibitors (adalimumab p<0.01, infliximab p=0.0003) and GCs (betamethasone and methylprednisolone at 1ug/ml p<0.01 and at 10ug/ml p<0.04) significantly reduced TNF levels in cultured media derived from SFMCs of PsA patients (n=8) compared to medium. None of the other biologics reduced TNF levels in culture (Fig. 1A). Additionally, %CD14+CD16+ SFMCs derived from PsA patients (n=11) were significantly reduced by TNF inhibitors (p=0.0003) compared to medium, however, other biologics and GCs did not display similar activity (Fig. 1B).Figure 1.Both TNF inhibitors and GCs block TNF secretion but exhibit different activity on inflammatory CD14+CD16+ monocytes derived from SFMCs of PsA patients. SFMCs were co-cultured for 7 days in the presence of adalimumab, infliximab, secukinumab and ustekinumab at 10ug/ml or with betamethasone and methylprednisolone at 1ug/ml and 10ug/ml. Medium alone was used as a control. (A) Culture supernatants were analyzed for TNF levels by ELISA (n=8). (B) Cells were analyzed for %CD14+CD16+ monocytes by flow cytometry (n=11). All p values were calculated by the non-parametric one-way ANOVA Kruskal-Wallis test and Dunn’s multiple comparison test, *p<0.04, **p<0.01 and ***p=0.0003.Conclusion:Our data demonstrated marked activity mediated by TNF inhibitors in comparison with other biologics tested for their ability to suppress TNF secretion and inflammatory CD14+CD16+ monocytes. In contrast, GCs suppressed TNF secretion but did not significantly change the proportion of inflammatory CD14+CD16+ monocytes. These findings suggest an additional mechanism of action exerted directly by TNF inhibitors on synovial monocytes and which differs from that of GCs. These results warrant further studies of the therapeutic potential of local peripheral activity of TNF inhibitors for clinical application.Reference:[1]Carubbi F, Zugaro L, Cipriani P, Conchiglia A, Gregori L, Danniballe C, et al. Safety and efficacy of intra-articular anti-tumor necrosis factor alpha agents compared to corticosteroids in a treat-to-target strategy in patients with inflammatory arthritis and monoarthritis flare. Int J Immunopathol Pharmacol. 2016;29:252-66.Disclosure of Interests:None declared

scholarly journals Synovial effusion and synovial fluid biomarkers in psoriatic arthritis to assess intraarticular tumor necrosis factor-α blockade in the knee joint

2010 ◽  
Vol 12 (4) ◽  
pp. R148 ◽  
Author(s):  
Ugo Fiocco ◽  
Paolo Sfriso ◽  
Francesca Oliviero ◽  
Pascale Roux-Lombard ◽  
Elena Scagliori ◽  
...  
Keyword(s):  
Psoriatic Arthritis ◽  
Tumor Necrosis Factor ◽  
Synovial Fluid ◽  
Knee Joint ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Synovial Effusion ◽  
Factor Α ◽  
Necrosis Factor

scholarly journals Cytokines regulate HIV-1 replication and lymphocyte apoptosis in vitro

2013 ◽  
Vol 94 (6) ◽  
pp. 906-910 ◽  
Author(s):  
S V Boichuk ◽  
P D Dunaev ◽  
I G Mustafin
Keyword(s):  
Flow Cytometry ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis Factor Alpha ◽  
Tumor Necrosis ◽  
Lymphocyte Apoptosis ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Hiv 1 ◽  
Necrosis Factor

Aim. To study the ability of cytokines - interleukin-2, interleukin-7 and tumor necrosis factor alpha to induce human immunodeficiency virus type 1 (HIV-1) replication and lymphocyte apoptosis in vitro. Methods. Peripheral blood mononuclears were separated by centrifugation on a ficoll paque solution specific density gradient. Lymphocytes were cultivated in RPMI 1640 medium with addition of L-glutamine, embryonal bovine serum, antibiotics and cytokines (interleukines-2, -4, -7, tumor necrosis factor alpha). To infect the lymphocytes, a laboratory strain of HIV-1 NL4-3 (NIH ResReag. Prog., USA) was used. HIV-1 replication was assessed by р24gag viral protein level in culture supernatants (ELISA) and its cytozolic level in lymphocytes (flow cytometry). Lymphocyte apoptosis was assessed by flow cytometry using the following parameters: (1) decrease of transmembrane mitochondrial potential; (2) increase in phosphatidyl serine molecules expression. Lymphocyte activation was assessed by CD25 and HLA-DR molecules expression (flow cytometry). Results. Cytokines induce the HIV-1 replication in lymphocytes in vitro. HIV-1 replication was noted only if inactivated lymphocytes were present in the culture. At the same time, lymphocytes not expressing the classical activation markers (CD25 and HLA-DR) were present among the lymphocytes producing HIV-1 indicating the possible alternative mechanism of HIV-1 replication, not dependent on cell activation. This fact might also be an evidence of viral replication processes in the pool of latently-infected lymphocytes, not expressing the classic activation markers. The abovementioned cytokines promote apoptotic death of uninfected lymphocytes in vitro, backing up the infected cells viability and thus promoting HIV-1 replication. Conclusion. Cytokines (interleukines-2, -4, -7, tumor necrosis factor alpha) which are known as factors supporting the immune system homeostasis and immune response formation, might also play a negative role in HIV-1 pathogenesis - induce HIV-1 replication in lymphocytes and, probably, lead to reactivation of the pool of latently-infected lymphocytes, deepening the lymphopenia and leading to disease progression.

scholarly journals Directs effects of erythropoietin on functional properties of human monocytes/macrophages

2018 ◽  
pp. 26-33
Author(s):  
О.Б. Мелащенко ◽  
М.Е. Меняйло ◽  
В.В. Малащенко ◽  
Н.Д. Газатова ◽  
А.Г. Гончаров ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Interleukin 4 ◽  
Human Monocytes ◽  
Membrane Expression ◽  
Flow Cytofluorometry ◽  
Necrosis Factor

Цель. Исследовали прямые эффекты эритропоэтина (erythropoietin, Epo) на функциональную активность моноцитов/макрофагов (Мц/Мф) человека in vitro. Методы. Популяцию CD14 клеток получали из мононуклеарных клеток (МНК) крови человека методом позитивной магнитной колоночной сепарации. Мц/Мф культивировали без липополисахарида (ЛПС) или с ЛПС в течение 24 ч. Мембранную экспрессию CD14 ( гликозилфосфатидилинозитол-связанный белок), CD16 (низкоафинный Fc-рецептор), CD119 (рецептор интерферона-g), СD124 (рецептор интерлейкина-4) и CD197 (хемокиновый рецептор CCR7) оценивали методом проточной цитофлюорометрии. Содержание фактора некроза опухоли-a (tumor necrosis factor-a, TNF-a), интерлейкина-1b (interleukin-1b, IL-1b), IL-6 и IL-10 в культуральных супернатантах определяли иммуноферментным методом. Результаты. Показано, что Epo достоверно снижал количество CD14, CD124 и CD197 клеток, но не CD16 клеток среди неактивированных Мф. Epo также заметно уменьшал количество CD197 клеток, но не СD14, CD124 и CD197 клеток, среди Мф, активированных ЛПС. Кроме того, Epo был способен умеренно усиливать продукцию интерлейкина-1b (interleukin-1b, IL-1b) и IL-6, неактивированными Мф и, наоборот, снижать продукцию этих цитокинов, осуществляемую активированными Мф. В то же время, Epo не оказывал существенного влияния на макрофагальную продукцию TNF-a и IL-10. Заключение. Направленность и выраженность регуляторных эффектов Epo на функции Мц/Мф зависят от активационного состояния этих клеток. Aim. We studied direct effects of erythropoietin (Epo) on the function of human monocytes/macrophages (Mc/Mphs) in vitro. Methods. CD14-positive cells were isolated from human peripheral blood mononuclear cells (PBMCs) by positive magnetic separation. Mc/Mphs were cultured without or with lipopolysaccharide (LPS) for 24 hours. Membrane expression of CD14 (membrane glycosylphosphatidylinositol-bound protein), CD16 (low-affinity Fc receptor), CD119 (interferon-g receptor), CD124 (interleukin-4 receptor), and CD197 (chemokine receptor CCR7) was evaluated by flow cytofluorometry. The content of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), IL-6 and IL-10 in culture supernatants was determined by the enzyme immunoassay. Results. Epo was found to significantly reduce the amount of CD14, CD124 and CD197 cells, but not CD16 and СD119 cells, among non-activated Mphs. Epo also detectably reduced the content of CD197 cells, but not CD14, CD124 and CD197 cells, among LPS-activated Mphs. In addition, Epo was able to moderately increase the production of interleukin-1b (IL-1b) and IL-6 by non-activated Mphs and, vice versa, to reduce the production of these cytokines by activated Mphs. At the same time, Epo did not significantly affect the Mph production of TNF-a and IL-10. Conclusion. Direction and intensity of regulatory effects of Epo on Mphs functions depend on the activation status of these cells.

scholarly journals Evaluation of Th9 lymphocytes in peripheral blood of rheumatoid arthritis patients and correlation with anti-tumor necrosis factor therapy: results from an in vitro pivotal study

Reumatismo ◽  
2016 ◽  
Vol 68 (2) ◽  
pp. 83 ◽  
Author(s):  
R. Talotta ◽  
A. Berzi ◽  
F. Atzeni ◽  
D. Dell'Acqua ◽  
P. Sarzi Puttini ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Tumor Necrosis Factor ◽  
Peripheral Blood ◽  
Tumor Necrosis ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
T Helper ◽  
Th9 Cells ◽  
Necrosis Factor

The aim of this study was to determine the prevalence of T helper 9 (Th9) lymphocytes in rheumatoid arthritis (RA) patients and to identify a possible association between the percentage of Th9 and the discontinuation of a biological treatment with an anti-tumor necrosis factor (TNF) (infliximab). We collected peripheral blood mononuclear cells (PBMCs) from 55 consecutive RA outpatients and 10 healthy controls. Among RA patients, 15 were not receiving any immunosuppressive drug, 20 were successfully treated with infliximab and 20 discontinued infliximab because of adverse events or inefficacy and were treated with other biological agents. PBMCs were cultured with/without infliximab 50 mg/L for 18 h, and the percentage of Th9 cells was assessed by means of flow cytometry. Th9 lymphocytes were identified as interferon gamma, interleukin (IL)4-, IL17-, IL9-secreting cluster of differentiation 4 (CD4)+ T cells. Cytometric analysis revealed no significant decrease in the percentage of Th9 cells after infliximab exposure in any of the groups, although it was lower in healthy controls than RA patients either before and after the infliximab stimulation assay. Th9 cells are IL-9-secreting T helper lymphocytes whose role in RA is still poorly known. IL-9 levels are increased in RA patients, in whom this cytokine plays a crucial role. Th9 cells are the major producers of IL-9, and their prevalence is higher in RA patients than in healthy subjects; however our experiment <em>in vitro</em> does not demonstrate an association between Th9 lymphocytes and the response to infliximab. Further studies are required to evaluate the real involvement of Th9 population in the immunogenicity of anti-TNF agents.

scholarly journals Three-Color Flow Cytometry Detection of Intracellular Cytokines in Peripheral Blood Mononuclear Cells: Comparative Analysis of Phorbol Myristate Acetate-Ionomycin and Phytohemagglutinin Stimulation

2001 ◽  
Vol 8 (2) ◽  
pp. 303-313 ◽  
Author(s):  
Jarołsaw Baran ◽  
Danuta Kowalczyk ◽  
Mariola Oz˙óg ◽  
Marek Zembala
Keyword(s):  
Flow Cytometry ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Mononuclear Cells ◽  
Myristate Acetate ◽  
Peripheral Blood Mononuclear ◽  
Color Flow ◽  
Intracellular Cytokines ◽  
Blood Mononuclear Cells ◽  
Necrosis Factor

ABSTRACT The assessment of intracellular cytokines at the single-cell level by flow cytometry has recently become a potent tool in many areas of cell biology and in defining the role of cytokines in various human diseases. Three-color flow cytometry for detection of intracellular cytokines combined with simultaneous determination of lymphocytes (CD3+ and CD4+) or monocytes (CD33+and CD14+) was used for comparison of phytohemagglutinin (PHA)-and phorbol myristate acetate (PMA)-ionomycin-induced production of intracellular cytokines in peripheral blood mononuclear cells (PBMCs) of healthy donors. We found that the number of PBMCs stained for tumor necrosis factor alpha and gamma interferon after 6 h of activation was higher when PMA-ionomycin was used for stimulation, while the frequencies of cells positive for interleukin 4 (IL-4) were similar for both stimulators. However, PMA-ionomycin stimulation caused prominent alterations of cell morphology and membrane expression of CD4 and CD14. In contrast, PHA did not cause downregulation of surface markers and resulted in less pronounced alterations in both forward and side scatter signals during flow cytometry analysis. Moreover, during 48 h of culture PHA stimulated tumor necrosis factor beta and IL-10 production, which was not observed when PMA-ionomycin was used. We conclude that the use of PHA for cell activation may limit in vitro artifacts and allow more precise analysis of intracellular cytokine production in various disease states.

Altered interleukin-1 and tumor necrosis factor production and secretion during pyrogenic tolerance to LPS in rabbits

1994 ◽  
Vol 267 (1) ◽  
pp. R329-R336 ◽  
Author(s):  
G. Wakabayashi ◽  
J. G. Cannon ◽  
J. A. Gelfand ◽  
B. D. Clark ◽  
K. Aiura ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Mononuclear Cells ◽  
Interleukin 1 ◽  
Interleukin 1 Beta ◽  
Peripheral Blood Mononuclear ◽  
In Vitro Synthesis ◽  
Circulating Levels ◽  
Necrosis Factor

Rabbits were injected intravenously with 10 micrograms/kg of endotoxin [lipopolysaccharide (LPS)] on days 0, 1, and 7, and rectal temperatures were monitored. The febrile responses were compared with circulating levels of interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) and in vitro synthesis of these cytokines by peripheral blood mononuclear cells (PBMC) isolated just before the injection of LPS. Fever after the first LPS injection was biphasic on day 0, attenuated and monophasic after the second LPS injection on day 1, and augmented after third injection of LPS on day 7. On day 1, circulating TNF and IL-1 beta levels were significantly (P < 0.05) decreased compared with those on days 0 and 7. Similarly, TNF and IL-1 beta synthesis by LPS-stimulated PBMC were significantly reduced on day 1. On day 7, cellular synthesis and secretion of IL-1 beta were significantly increased compared with that on day 0. A significant positive correlation was observed between fever index and total in vitro IL-1 beta synthesis by LPS-stimulated PBMC (r = 0.866, P = 0.001). These data demonstrate that pyrogenic tolerance in the rabbit after a single LPS injection is associated with decreased circulating IL-1 beta and TNF levels as well as decreased production of these cytokines in vitro. In addition, the pyrogenic hyperresponsiveness to LPS after 7 days is associated with increased synthesis and secretion of IL-1 beta from PBMC in vitro.

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