RU2590696C2 - Agent for differentiation of monocyte-like cells - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: described is a lipopolysaccharide, endotoxins antagonist, produced strain Rhodobacter capsulatus PG, which are deposited in RAS IBFM under number B-2381 D, as an agent for monocyte-like cell differentiation. Presented lipopolysaccharide Rhodobacter capsulatus PG is physiological, practically nontoxic. Invention can be used as an agent for monocyte-like cell differentiation.

EFFECT: proposed device can be used for preparing cardiologic drugs, particularly to oncology.

1 cl, 3 dwg, 2 tbl

Description

The invention relates to microbiology, biotechnology, pharmacology and medicine and can be used to differentiate promonocytes and monocytes in clinical and experimental medicine.

Promonocytic human leukemia cell lines (U937 and THP-1) are widely used as model systems for studying differentiation mechanisms. Differentiation is characterized by a change in various cellular parameters of cells compared with the corresponding undifferentiated phenotype. Such changes relate to morphology, metabolic processes and cell growth. U937 and THP-1 cells represent various stages of monocyte development [14].

There are various agents that can induce the differentiation of cells of these lines into mature monocytes. Agents such as dimethyl sulfoxide (DMSO) [7], retinoic acid [9], vitamin D3 [10], cytokines [3, 13] and phorbol esters, in particular 12-O-tetradecanoylphorbol-13-acetate (TPA) [ 4], can contribute to the differentiation of immature monocytes into monocytes / macrophages.

The condition for differentiation into mature macrophages is an increase in adhesion to the surface of the culture plates and, as a result, a sharp change in the shape of the cell. Undifferentiated U937 and THP-1 cells have a rounded shape and grow in suspension. Both types of cells have a small cytoplasm and a large nucleus. After differentiation with TPA, most cells attach to the culture dies. For THP-1 cells, aggregate formation is especially characteristic due to the development of intercellular pseudopodia [11].

CD14, originally described as a monocyte differentiation marker, is a 55 kDa glycoprotein. Extensive studies have shown that CD14 is involved in mediating responses to several bacterial membrane products, especially bacterial LPS. CD14 is not detected on the surface of precursor monocytes and increases dramatically during their differentiation into monocytes. Thus, surface expression of CD14 is an excellent model for studying the mechanisms of myeloid cell maturation [12].

CR3 (β ₂ -integrin) is another classic marker of monocytic differentiation involved in cell adhesion and also functioning as a complement receptor. CD11b, a CR3 subunit, is a glycoprotein (160 kDa mass) that binds non-covalently to the β ₂ subunit of the CD18 partner. The CD11b / CD18 complex is expressed on mature myeloid cells and is widely used as an early marker of monocyte differentiation [5].

Retinoic acid (RA) is an active form of retinol (Vitamin A). RA, being a physiological agent, is able to form bonds with nuclear receptors, by which it triggers a cell response. It should be noted that almost all body cells are sensitive to retinol.

The growth of THP-1 cells is inhibited after the addition of RA at a concentration of 10 ^-6 M. In addition to stopping the growth, morphological changes occur: the cells acquire an irregular shape and lobular nuclei. In addition to morphology, differentiation of THP-1 cells using RA leads to other changes. The phagocytic activity noticeably increases. However, RA does not cause an increase in adhesive ability [6].

1α, 25-dihydroxyvitamin D ₃ (VD ₃ ), like retinoic acid, is a physiological agent and ligand for the superfamily of nuclear receptors. Easily penetrating cell and nuclear membranes (of the same type as RA), receptors of this type act directly on genes, playing the role of transcription factors that control gene expression.

The pleiotropic effects of VD _{3 are} mediated primarily through the intracellular vitamin D receptor (VDR). VDR is a member of the superfamily of nuclear steroids and retinoic acid receptors. Thus, it functions as a ligand-dependent transcription factor that regulates the activation of vitamin D target genes. At the molecular level, VDR activates its target genes by interacting with specific DNA sequences that exist as vitamin D response elements (VDRs) called "genomicaction" [5].

In attempts to determine the molecular mechanisms of regulation of cellular responses to VD ₃ , the possibility of the existence of alternative signaling pathways to the classical genome action program was considered. Indeed, over the past decade, experimental data for “non-genomic” signaling have the concept of exclusive VDR-mediated genomic action. For example, VD ₃ stimulates the rapid formation of secondary messengers, including ceramides, cAMP, inositol, the release of calcium cations, and activates various protein kinases, including protein kinase C, Raf, mitogen activated protein (MAP kinase), and the Src kinase family. However, the physiological significance of non-genomic signaling alone or with respect to VDR-mediated genomic action is still unclear. In addition, it is not known whether VD ₃ interacts with classic VDR for non-genomic signaling or whether an alternative receptor system is involved.

Treatment with VD ₃ increases the surface expression of CD11b and CD14 on HL-60, U937, and THP-1 cells [5]. VD ₃ causes an increase in phagocytic activity, recorded by the fluorescence of latex beads.

PMA phorbol ester (phorbol 12-myristate 13-acetate) is one of the most powerful non-physiological differentiation agents. PMA easily penetrates the cell and, due to its structural similarity to diacylglycerol (DAG), interacts with its binding site on the protein kinase C molecule (PKC) [8].

The expression of surface M-CSF receptors is one of the first events associated with the monocytic differentiation of leukemic cells into monocytes. Recent studies using several leukemia cell models have shown that the level of c-fms transcripts increases dramatically when they begin to undergo monocytic differentiation under the influence of PMA. It is believed that the activation of protein kinase C (PKC) plays a major regulatory role at the beginning of differentiation. In the normal physiological process, PKC is activated by endogenous diacylglycerol (DAG), the product of the hydrolysis of membrane phospholipids under the action of phospholipase C (PLC).

The effects caused by differentiation in THP-1 cells begin to manifest themselves upon treatment with PMA concentration of 10 nM: cell growth is inhibited, more than 80% of the cells become adhesive within 24 hours after exposure to PMA. After PMA treatment, an increase in the number of vacuoles is observed, and further incubation in the presence of this agent leads to an increase in size and lengthening of the shape, as in normal blood monocytes in culture. In addition, when THP-1 cells undergo PMA treatment, an increased level of c-fms transcripts is detected after 6-12 hours.

RMA molecules are relatively stable and have a long-term effect on THP-1 cells, which determines their long-term effect on the effects of physiological agents. However, PMA activates the cells so strongly that their further use as an object of study of the influence of lipopolysaccharides (endotoxins) is impossible [12].

The objective of the invention is a physiological, practically non-toxic agent for the differentiation of monocyte-like cells.

The problem is solved by a non-toxic lipopolysaccharide from the phototrophic bacterium Rhodobacter capsulatus PG.

It is previously known to use the strain Rhodobacter capsulatus PG as a producer of lipopolysaccharide, an endotoxin antagonist [1].

This strain of Rhodobacter capsulatus PG was deposited by the All-Russian Collection of Microorganisms of the IBPM RAS [2].

The ability of non-toxic LPS Rhodobacter capsulatus PG to differentiate the cells of the THP-1 monocytic line into monocytes and macrophages was studied. The differentiating activity of LPS was studied by expression of surface receptors (TLR4, CD11b and CD14) and changes in the morphological characteristics of THP-1 cells.

1. A study of the effect of LPS Rhodobacter capsulatus PG in the range of 100-1000 ng / ml on the morphology of THP-1 cells.

The object of the study was the THP-1 monocytic cell line from the ATCC ^® TIB ™ 202 collection (USA). THP-1 cells were cultured in RPMI 1640 medium (Sigma) containing 2 mmol / L L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, (L-Glutamin-penicillin-streptomycin solution, Sigma) and 10% inactivated bovine serum (Hyclon) in 25 cm ² , 50 ml culture bottles (Orange Scientific) in a CO ₂ incubator (Jouan, France) at 37 ° C and 5% CO ₂ . Viability, determined by the staining of cells with trypan blue, was 88-95%.

To a suspension of THP-1 cells in RPMI 1640 medium (2 × 10 ⁶ cells / ml) was added an aqueous LPS solution of R. capsulatus PG at a final concentration of 100 or 1000 ng / ml and incubated for 24 hours in a CO ₂ incubator at 37 ° C and 5% CO ₂ .

To assess the degree of differentiation, a visual analysis of THP-1 cells was used after 24-hour incubation with LPS R. capsulatus PG. For this purpose, the proportion (in% of the total number of cells in 7–8 full fields of view) of cells with altered morphology was determined, in particular, having noticeable pseudopodia, increased sizes, and fusiform or elongated cells. The normal morphology of undifferentiated THP-1 cells is rounded cells without noticeable pseudopodia. The results of the visual analysis are presented in table 1 and in FIG. 1, 2a, 2b, 2c.

In FIG. 1. presents the change in the morphology of THP-1 cells after 24 hours of exposure with LPS R. capsulatus PG (* - differences from the control are significant at p <0.05).

In FIG. 2a shows a change in the morphology of THP-1 cells under the action of LPS R. capsulatus PG (magnification 40 × 1.5 × 10, no staining), control.

In FIG. 2b shows the change in the morphology of THP-1 cells under the action of LPS R. capsulatus PG (magnification 40 × 1.5 × 10, no staining), cells after stimulation of LPS R. capsulatus PG 100 ng / ml.

In FIG. 2c shows the change in the morphology of THP-1 cells under the action of LPS R. capsulatus PG (40 × 1.5 × 10 magnification, no staining), cells after stimulation of LPS R. capsulatus PG 1000 ng / ml.

The proportion of THP-1 cells with altered morphology was in the control variant (25.2 ± 2.4)%. From those presented in table 1 and in FIG. 1, 2 (a, b, c) of the results, it can be seen that the LPS of R. capsulatus PG, introduced at the minimum dose (100 ng / ml), did not cause a significant change in the morphology of THP-1 cells (the proportion of cells with altered morphology is 23.9 ± 1.8% compared with control). Therefore, introduced in this concentration of LPS R. capsulatus PG does not have a biological effect on the change in the morphology of THP-1 cells.

LPS of R. capsulatus PG at a concentration of 1000 ng / ml caused a significant increase in the proportion of altered THP-1 cells to (37.4 ± 4.3) pg / ml. This value significantly differs from the control (25.2 ± 2.4)% and indicates the ability of LPS from R. capsulatus PG to cause a change in the morphology of THP-1 cells and, possibly, increase the degree of their differentiation.

2. Search for the optimal LPS concentration of Rhodobacter capsulatus PG in the range of 100-1000 ng / ml for a change in the level of surface receptors (TLR4, CD11b and CD14) of undifferentiated THP-1 cells.

We used antibodies to TLR4, CD14 and CD11b receptors labeled with a fluorescent label: Alexa Fluor 488 anti-human CD14 (Clone HCD14), Alexa Fluor 488 anti-human CD11b (Clone ICRF44) (BioLegend); Alexa Fluor 488 anti-human CD284 (TLR4) (Clone HTA 125) (eBioscience).

After incubation of the cells with LPS R. capsulatus PG at a final concentration of 500 or 1000 ng / ml for 24 hours in a CO ₂ incubator at 37 ° C and 5% CO ₂ , THP-1 cells were separated from the culture medium by centrifugation and resuspended in staining buffer (Cell Staining Buffer, BioLegend) in the calculation of 10 ⁵ -10 ⁶ cells in 100 μl of buffer.

To exclude non-specific binding of antibodies to the studied receptors, 5 μl of Fc receptor blocking buffer (Human Tru Stain FcX ™, BioLegend) was added to each sample and incubated at room temperature for 10 minutes. Then, 5 μl of a solution of the corresponding antibodies were added to the samples and incubated for 30 min at 4 ° C. Then, Cell Staining Buffer (2 ml each) was washed 2 times, resuspended in 400 μl of the same buffer and transferred to a cytofluorimetric tube.

Samples were analyzed using an EPICS XL-MCL flow cytometer (Beckman Coulter, USA). The level of surface reception of THP-1 cells was evaluated by the average fluorescence intensity (MnIX) of the sample. 6000 cells were counted in each sample.

FIG. 3. Change in the expression level of TLR4, CD11b and CD14 receptors under the influence of LPS R. capsulatus PG in%. Presents the average values of changes in the level of expression of receptors (n = 3) and the standard error in percent (%) relative to the control, taken as 100%.

From those presented in table 2 and in FIG. The 3 results show that the LPS of R. capsulatus PG most significantly affects the expression of CD11b receptors. Of the studied concentrations, LPS R. capsulatus PG, introduced at a concentration of 500 ng / ml, has the greatest influence on the change in the level of surface receptors.

Based on the results on the change in the morphology of THP-1 cells and the expression of TLR4, CD11b and CD 14 receptors on their surface under the influence of non-toxic LPS Rhodobacter capsulatus PG, it follows that non-toxic LPS Rhodobacter capsulatus PG has a maximum differentiating activity at a concentration of 500 ng / ml.

The proposed tool can find application in the creation of drugs, in particular for oncology.

Bibliography

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2. Certificate of deposit of the strain Rhodobacter capsulatus PG. Registration number VKM V-2381 D, 12/21/2005.

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Claims (1)

A lipopolysaccharide, an endotoxin antagonist produced by the strain Rhodobacter capsulatus PG, deposited in VKM IBPM RAS under the number B-2381 D, as a means for the differentiation of monocyte-like cells.

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