SU1730594A1 - Method of interleukin-1 determination in human monocytes - Google Patents

Abstract

Use: The invention relates to medicine, primarily immunology, and can be used in the diagnosis of the state of the immune system. The purpose of the invention is to simplify, accelerate and improve the accuracy of the determination of IL-1 human monocytes. After separation from lymphocytes in plastic wells, blood monocytes are washed by adhesion, activated with lipopolysaccharide for 1.5-3 hours, washed again and an equal volume (0.2 mm) of autologous lymphocyte suspensions and phytohemagglutinin is added to the wells in a suboptimal dose and after cultivation, as compared with the control samples, the index of recovery of the reaction of blasttranspor, mation is calculated. The positive effect of the invention is to simplify and accelerate the determination of IL-1 by pulsed activation (1.5-3 hours) of monocytes by lipopolysaccharide and using an autologous system of immunocompetent cells (monocytes and lymphocytes). 2 Il. 1 tab.

Description

The invention of otnostis to medicine, mainly to immunology, and can be used in the diagnosis of the state of the immune system.

The purpose of the invention is to simplify, accelerate and improve the accuracy of the determination of IL-1 human monocytes.

The goal is achieved by clearing blood monocytes after separation from lymphocytes in plastic wells by adhesion, activating with lipopolysaccharide for 1.5-3 hours, re-washing and adding equal volume (0.2 ml) of autologous lymphocytes and phytohemagglutinin to the wells and after cultivating the mixture of cells, the index of recovery of the blast transformation is calculated, as compared with the control samples.

The method is carried out as follows.

Heparinized (20 U), venous blood in a volume of 5-6 ml is layered on a 2 ml Ficoll-Gipak gradient (1.077 g / cm3) in a centrifuge tube and centrifuged at 400 g for 40 minutes. The mononuclear fraction in the interphase layer is collected, washed three times with physiological saline, and the cell suspension is prepared to a concentration of 2 x 106 in 1 ml of RRM1 -1640 medium with 10% inactivated AB (IV) serum. To separate adherent and non-adherent cells, a suspension of mononuclear cells is poured into 0.2 ml per well in plastic flat-bottomed plates for cell culture and incubated for 45 minutes at 37 ° C and 5% C02. Non-adherent cells consisting of 95% lymphocytes (azure-eosin stain)

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collect and store at 4 ° C until use. Adherent cells consisting of 92% monocytes (staining for nonspecific esterase) are gently washed several times with medium 199 and 0.1 ml of PPM1-1640 medium is added to all wells. Cells are activated by lipopolysaccharide E. Coli (LPS Sen / a) by the pulsed method. To this end, 0.02 ml (20 µg / ml) of LPS was added to a portion of the wells with monocytes, 0.02 ml of RPM1-1640 medium was added to others and incubated at 37 ° C and 5% C02 at different time intervals (0, 5; 1.5; 3.0 hours). At the end, all supernatant liquid is removed and the wells with cells are gently washed three times with medium 199. Then, 0.2 ml of autologous non-adherent cells (lymphocytes) in a concentration of 2x10 cells is added to a monolayer of monocytes (activated and non-activated LPS). RPM1-1640 medium with 20 mM glutamine, 10% inactivated AV (IV) serum and 50 μg / ml gentamicin. A suboptimal dose (1 µg / ml) F HA in a volume of 0.02 ml is added to a portion of the wells (test) with cell mixtures consisting of activated or intact monocytes and lymphocytes. An equal volume of RPM1-1640 medium is added to the other (control). Plates are placed in a thermostat at 37 ° C with 5% COa and cultured for 72 hours. At the end of the reaction, lymphocyte blast-transformation (RBTL) prepare cell smears in which blast cells.

RBTLs with the original mononuclear cells and with non-adherent cells (lymphocytes) are set to control the spontaneous and mitogen-induced proliferative response of the cells. For this, 0.2 ml of a suspension of cells with a concentration of 2x10 per 1 ml of RPM1-1640 medium with 20 mM glutamine, 10% inactivated AB (IV) serum and 50 μg / ml gentamicin are placed in the wells. 0.02 ml of PHA (10 µg / ml) is added to the test wells, the same amount of medium is added to the control wells and cultured at 37 ° C with 5% C02 for 72 hours, followed by blast cell determination. RBTL in all studies was assessed by the activation index (AI) —the ratio of the number of blast cells in cultures treated with PHA (experiment) to the number of blast cells in cultures not treated with PHA (control).

To assess the production of IL-1 by monocytes, the blast transformation reaction recovery index (CRRI) is used.

Lymphocytes + monocytes activated by the AI by the pulsed method, or intact monocytes - AI (lymphocytes)

IA (mononuclear cells) IA (lymphocytes)

In terms of research, IL-1 was determined in daily monocyte supernatants activated by LPS by the pulsed method, as well as in intact monocyte daily supernatants according to the invention. For this, the cell monolayer is incubated with or without LPS for 0.1; 1.5; 3 hours. Then they are gently washed three times with medium 199 from the inductor, suspended in RPM1-1634 medium, and cultured for 1 day at 37 ° C with 5%. Then, the collected supernatants are centrifuged at 400d15 min and the IL-1 activity is determined in them. The data obtained is expressed in the IIRB.

IA (lymphocytes) + IL) - IA (lymphocytes)

IA (mononuclear cells) - IA (lymphocytes)

where IL is the daily supernatants of monocytes activated by the pulsed LPS or intact monocytes.

The results obtained are statistically processed using Student’s criteria.

Results. Intact monocytes stimulate the proliferative response of autologous lymphocytes in response to a suboptimal dose of PHA compared with the proliferation of lymphocytes alone (figure 1). However, these cells are not able to restore the proliferation of lymphocytes to the values obtained in RBTL with mononuclear cells, since the IBRB is equal to 0.6 ± 0.09. After activation of LPS monocytes for 0.5 hours, their ability to stimulate lymphocyte proliferation is not significantly different from the action of intact cells (Fig. 1). The proliferative response of lymphocytes increases significantly (P 0.05) after activation of LPS monocytes for 1.5 hours and saved when using monocytes activated by LPS for 3 hours (figure 1). The findings suggest that after pulsed activation of LPS, monocytes acquire the ability to enhance the proliferative response of autologous lymphocytes in response to a suboptimal dose of PHA.

LPS is a good inducer of IL-1 production by monocytes. Therefore, after pulsed activation of LPS, human monocytes are supposed to acquire the ability to produce IL-1, which stimulates the proliferation of autologous lymphocytes. To confirm this, IL-1 was determined in diurnal supernatants of monocytes activated by LPS using a pulsed method using a known method. In addition, IL-1 in daily supernatants of intact human monocytes was determined in a known manner. It turned out that the supernatants of intact cells stimulate lymphocyte proliferation in response to a suboptimal dose of PHA. IBRBVetihlu x equals 0.92 ± 0.12 (figure 2), which can be seen from the results obtained according to a known method. An increase in IVRB is observed when using monocyte supernatants activated by LPS in a pulsed manner for 1.5 and 3 hours (Fig. 2), while monocytes supernatants activated by LPS for 0.5 h do not have this effect (Fig. 2 ).

The results of these studies indicate that monocytes activated by LPS using the pulsed method acquire the ability to produce IL-1, which is determined in the daily supernatants of these cells using a known method.

Thus, the data show that monocytes activated by LPS using the pulsed method produce IL-1, which is determined by stimulating the proliferation of autologous lymphocytes in response to a suboptimal dose of PHA.

PRI me R 1. Studies were conducted on blood from 25 donors of a blood transfusion station. Statistically processed results are presented in figure 1. After activation of LPS monocytes for 0.5 hours, their ability to stimulate the proliferation of autologous lymphocytes in response to a suboptimal dose of PHA does not differ significantly from the action of intact monocytes. This indicates that in both cases the same ability of human monocytes to produce IL-1 is determined. When monocytes of LPS are activated for 1.5 and 3 hours, the proliferative response of lymphocytes to the suboptimal concentration of PHA increases (05). Based on the obtained data, we can assume that human monocytes activated by LPS are pulsed

method (1.5-3 hours), produce IL-1, which is determined by its ability to stimulate the proliferation of autologous lymphocytes, stimulated by a suboptimal dose of PHA.

Example 2: IL-1 determination of human monocytes was carried out using known and proposed methods in 30 donors of a blood transfusion station.

The data presented in the table.

When using a known method, the same amount of IL-1 was determined (IVRB 0.92 ± 0.12). When determining IL-1 of human monocytes by the proposed method, the IBRB is equal to 1.35 ± 0.1, which is higher than the data by a known method. Therefore, the proposed method improves the accuracy of the determination of IL-1 human monocytes.

The positive effect of the invention is to simplify, speed up and increase the accuracy of the determination of IL-1 of human monocytes by pulsed activation of monocytes by lipopolysaccharide and

use of the autologous system of immunocompetent cells (monocytes and lymphocytes).

Claims (1)

Claim method for determining interleukin-1 human monocytes by assessing the effect of monocyte culture products on the proliferative activity of PHA-stimulated human lymphocyte culture, characterized in that simplifying, speeding up and improving the accuracy of the determination, blood monocodes after separation from lymphocytes in plastic wells are washed by adhesion, activated by lipopolysaccharide for 1.5 - 3 hours, washed again and added to the wells in a volume of 0.2 ml of autologous lymphocyte suspension with phytohemagglutinin and, after cultivation, the index of the blast transformation reaction is evaluated, as compared to the control samples. UBPB W 7.2 1.0 0.8 0.6 DM 02 Q -Monocytes, actirodata NIC pulse method Shch-Tsntaktny monocytes 0.5 $ 1, 0 burden of sctiostion. tonocyte.poo ALS (hp, ls1) fig, 1 R BimPXffl ™ onocyto8 vmpio uanoi uMnuAhT