JPS63123394A - Differentiation-inducing factor - Google Patents

Abstract

PURPOSE:To obtain a differentiation-inducing factor which consists of protein derived from human cell, having specific molecular weight and differentiates human macrophage precursor cells into macrophage. CONSTITUTION:In the process of study of differentiation of myeloid cell strain, it is elucidated that a myeloid cell which is differentiated into a macrophage-like cell by 12-0-tetradecanoylphorbol-13-acetate produces differentiation-inducing factor of protein and purification and isolation thereof were successful. The differentiation-inducing factor is protein having 67,000+ or -5,000mol.wt. and is capable of differentiating human macrophage precursor cells such as U937, HL-60, etc., which are cell strains established as myelocytic leukemia cell capable of carrying out differentiation induction, into macrophage.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to factors that induce macrophage differentiation.

(Prior art) Macrophages are mononuclear phagocytes that play the most important role in the body's defense mechanism, including phagocytosis of foreign bacteria, damage to abnormal cells and tumor cells, and antigen presentation in the development of immunity. It is a cell that fulfills its purpose.

In the bone marrow, a portion of myeloid stem cells differentiate into monocytes, and protein differentiation factors that are transported to various tissues in the body in the bloodstream include intercellular leukokin-j (IL-j), granular -Macrophage colony formation stimulating factor (GM-OBF)
, macrophage colony formation stimulating factor (M-OBF), and gamma interferon (IFii-r), whose structures and properties are being clarified at the protein chemical or genetic level (proteins, nucleic acids,
Enzyme #Jl(z), ! 10(/9a'+)).

The molecular weight of these factors is IL-J is 2 ooo, '
GM-O8F is -4000, M-08F is 143. OO
Q.

IFN-r is xo, ooo-7! It is known that it is xooo.

In addition, many factors have been reported that have been predicted to exist but have not yet been purified as a single substance, and whose structures and properties are unclear1. Factors that occur (Japanese Patent Application Laid-Open No. 17-17209A), D of Sonson et al.
IF (J, Natl.

cancer, engineering, &7. /u, 2j ('J'
/) ), Ohiao et al.'s factor (Proc, Na
tl, Acad, Sci, r, 2. /, 209('
rj)) has been reported.

Due to these factors, macrophage progenitor cells differentiate into macrophages, which leads to decreased proliferation ability, increased adhesion to culture vessel walls, increased phagocytosis of microorganisms and dead cells,
Changes such as enhanced superoxide anion production ability, increased lysozyme enzyme activity, increased glucose uptake, increased receptors for immunoglobulin 10 and complement 03, and increased interleukin I production occur.

Therefore, the activity of the differentiation-inducing factor 1 can be measured using the degree of these changes as an index.

Such differentiation-inducing factors can be used to treat leukemia by inducing differentiation of myeloid leukemia cells, but they have also been shown to induce phagocytosis in adherent cells in human peripheral blood. Yo).

It is expected to be used as a therapeutic agent for tumors and infectious diseases, and it may also be used as a therapeutic agent for autoimmune diseases, as it helps the immune complex function in poor eating and digestion.

On the other hand, /J-0 is known as a non-protein differentiation-inducing factor, but in the process of studying the differentiation of myeloid cell lines, the present inventors found that myeloid cells differentiated into macrophage-like cells with TPA. The present invention was achieved by discovering that a novel proteinaceous differentiation-inducing factor was produced, and by purifying and isolating this.

(Structure of the Invention) That is, one gist of the present invention resides in a differentiation-inducing factor consisting of a protein with a molecular weight of 47.000±↓000 derived from human cells and capable of differentiating human macrophage progenitor cells into macrophages.

The present invention will be explained in detail below.

The differentiation-inducing factor (hereinafter abbreviated as "differentiation factor") in the present invention is derived from human cells and has a molecular weight of t'y, ooo± and O
U937 and HL, which are cell lines established as human myeloid leukemia cells, are the protein of OO.
By adding it to the culture medium of human macrophage progenitor cells (myeloid leukemia cells) such as -40, it reduces the proliferation ability of these cells, induces the ability to adhere to the culture vessel wall, and superoxide anion productivity, yeast oligophagy, yc
via receptors. It enhances phagocytosis, promotes differentiation factor regeneration M, increases expression of histocompatibility antigen 1, and increases production of proto-oncogene metsenger RNA. Maf
c1 The differentiation factor in the present invention is human peripheral blood leukocytes and
When it acts on bone marrow cells, it causes these cells to differentiate into macrophages, and exhibits phagocytosis and Fc receptor-mediated phagocytosis.
It causes cytotoxic activity that is not mediated by C receptors. But 1
Since the factor of the present invention itself does not have the activity of killing, for example, mouse L92 cells, it is concluded that the effect is different from tumor-killing factors such as lymphotoxin. The fact that it does not have an affinity for concanavalin A-Sepharose columns distinguishes it from other differentiation factors that have been overlooked so far, such as colony formation stimulating factors and interferons.

The differentiation factor in the present invention is 1, for example, monoblastic cell line U.
Human myeloid leukemia cells such as 937 were cultured in a medium such as RPMXlt≠Ob medium (manufactured by Jimeco) containing 2 to 20% fetal bovine serum to a cell number of 3 to IO x 10.
'Grow until it becomes /-, and this.

TPA so that o, o t - o, jμI/rat
After adding 1000 ml and continuing culturing for io-≠0 hours.

By changing to a serum-free medium such as DM140 medium (Kyokuto Pharmaceutical Co., Ltd.) and repeating serum-free culture for 20 to 60 hours, or after washing with a serum-free medium such as DM160,
It is produced in the culture supernatant by repeating 2o-go culture in RPuXtlao medium containing λ~-20X fetal bovine serum.

The culture supernatant containing the differentiation factor in the present invention is:

After concentration using an ultrafiltration membrane or ammonium sulfate salting out, DICAli
- Chromatography using anion exchangers such as Sephacel and Q-Sepharose (both manufactured by Pharmacia), hydrophobic chromatography using butyrut-iovar, phenyl, and Ipw (all manufactured by Toyo Soda), 78
By purifying in combination with gel filtration using K 0-2000 (manufactured by Toyo Soda Co., Ltd.) or the like, a protein showing a single band in 5Ds-X aerophoresis can be obtained.

In addition, as an acquisition method, for example, J) aDNA1! by genetic engineering method! It is also possible to clone the vector, insert it into an appropriate vector by a conventional method, and express it in E. coli, yeast, animal cells, etc.

The molecular weight of the differentiation factor thus obtained by 8DB-electrophoresis is A 7.000 thj, 000), and the first isoelectric point by first isoelectric focusing is gh, 2~! ,
− is. Moreover, its amino acid composition is shown in Table 1.

Table 7 Amino acid composition (shows the number of the relevant amino acids per 100 constituent amino acids. However, cysteine and tryptophan are excluded) Asparagine 10 Aspartic acid Ta~/1 Threonine! ~7 Serine μ~6 Glutamine + Glutamic acid /μ~/
6 Glycine 2~! alanine
6 ~ Tabarin
6~g Methionine OJ~/,! Isoleucine 1. ! ~3.0 Leucine
10N/, 2 tyrosine
3-≠Phenylalanine μ~6 Lysine 10~/λ Histidine
3~tA arginine
μ~! Proline ≠～6(
EXAMPLES The present invention will be specifically explained below using examples, but the present invention is not limited by the following examples unless the gist of the invention is exceeded.

Example +1) Culture method (/-/) Serum-free culture/D 10W1 was placed in a plastic petri dish containing % fetal bovine serum (manufactured by Armor Co., Ltd.), and the plate was heated to
Jil(7)TPA was added and incubated for cog hours. As a result, most of the fiU937 cells adhere to the plastic, so after removing the culture medium and dead cells with a pipette, aspirate.

With DM160 medium! The cells were washed d×/time and jm×/time, and further cultured in the same medium IO− for 1 hour. Culture supernatant liquid 7- was collected every λμ time.

Fresh M#O medium 7+dt- was added and culture continued. In this manner, cultured semen was collected to obtain cultured semen containing differentiation factors.

The activity of differentiation factor 1 was measured by measuring the ability to induce differentiation as shown in Test Example 1 described below. (The same applies to the following steps) (/-, ?) Culture with serum In the above (/-/), after washing with DM/40 medium, RPM/lg O medium/θ containing 10% fetal bovine serum again. - was added and the culture was continued, and the culture supernatant was collected every 7 d in the same manner as described above (/-/) to obtain a culture supernatant containing differentiation factors. This method uses the above (/-/
) 100 to 10 DD times more activity was obtained compared to the serum-free culture method.

(2) Purification method The culture soil liquid obtained as described above (/-/) is
,ooog, centrifuge for 20 minutes, and pass the supernatant through ultrafiltration membrane U.
It was concentrated using KjO (manufactured by Toyo Togami).

After concentration, iomM Tris-HCl, j% glycerin,
lIIQM dithiothreitol? Dialysis was performed against a buffer containing pH 7.-, followed by ion exchange chromatography using an anion exchanger Q-Sepharose (manufactured by Pharmacia). The column was equilibrated with the buffer described above, and elution was carried out in three stages using the buffer containing sodium chloride at 0 molar, o, iz molar, and 0.3 molar concentrations.

The differentiation factor (1) was eluted in the J molar fraction. Solid ammonium sulfate was added to the active fraction so that the final concentration was 1%,
After dissolution.

It was centrifuged for io, ooo, p, /o minutes, and the supernatant was mixed with phenyljPW (LKB glass bag) and the stiffening factor was ammonium sulfate concentration j! It was eluted in fraction A.

(3) Determination of molecular weight Both fractions containing differentiation factor 1 purified as described in (2) above were subjected to electrophoresis using a centrifugal concentration method using an ultrafiltration membrane ('7j)). That is, 20 μt was incubated at 100°C in sampling buffer containing 2-mercabutoethanol.
, boil for 3 minutes, and reduce the volume of this reaction solution to lOμti/mu! 2 was subjected to electrophoretic separation in an acrylamide gel. The electrophoresis is/
OmA de l! This was done by applying current for 1 minute, and then applying current at iOmA for 70 minutes. Gel after electrophoresis (i-5w1tze
r method (Anal, Biochem, Pf CoJ/(
'7ri)), silver staining was performed.

A band was observed only at the position corresponding to the molecular weight marker bovine serum albumin. or.

The same active fraction was added at pH 1 containing O1λ molar sodium chloride.
,, T equilibrated with 10 mmol phosphate buffer of male
Inject 200μ into the BK G-Koku00E'll column and turn λr On! When the absorption of the n-region and the elution of fractionation activity were examined, elution was found in both cases at a position consistent with that of Lye serum albumin.

Therefore, this differentiation factor has a molecular weight of approximately It was found to be a monomeric protein of tZOOO.

Test Example 1 Measurement of differentiation inducing ability / Culture solution containing lx10' U937 cells growing in suspension in RPM engineering/41/-0 medium containing OX fetal bovine serum / 00 #t aliquots were distributed into well plates. To this, the differentiation factor purified as in Example (2) above was added, and the mixture was cultured in Eupeta (37°C) in carbon dioxide gas.

Depending on the activity of the differentiation factors, the number of U937 cells that adhered to the bottom of the wells and matured increased.

When observed after a period of time, the liquid l-, which had an activity level of a differentiation factor that induced adhesion ability in J-0% cells, was placed in the l-position.

Further culturing was continued, and an anti-sheep red blood cell rabbit antibody (Japan Institute for Immunology) was added to the culture medium containing the attached U937aI cells.
Sheep red blood cells (8RBO) treated with 1 were given and observed under a phase contrast microscope.

After the culture≠g waiting period, all cells exhibited Fc receptor-mediated phagocytosis.

Also, j X / 0” ce1) s/ml of U93
Add 1 differentiation factor to 7 cell culture medium, and after culturing for 3 days Hat
The method of tOri et al. (J, 01 in, Invas°C, 7
2.237 ('13)) jj) NET staining was performed to measure the superoxide anion production ability, which increased depending on the amount of the added molecular factor.

Test Example λ Activity of inducing differentiation of macrophages from human terminal leukocytes or human bone marrow cells. Total leukocytes were separated from human terminal blood by density gradient centrifugation using Ficoll-no-ipack, and λ cycles were performed. PBE
After washing with I, the cells were suspended in a medium and used in experiments.

Bone marrow cells are obtained from patients without the bone marrow cell defect, as is the case with terminal blood.

Leukocytes obtained by separation by density gradient centrifugation were used in the experiment.

, distribute 2X/Os cells (terminal blood) or /X/06 cells (myeloid blood cells) into a 2-hole plate.

/d medium and! U differentiation factor was added and cultured at 37°C in a carbon dioxide gas incubator.

In the case of fine white blood cells, culture for 7 days.

Adhesive cells appeared even when differentiation factors were added.

In the case of addition of differentiation factors, the majority of adherent cells appear between 4 and 4 hours after the start of the experiment.

In the end, the number of adherent cells decreased compared to the case without additives! It was double that. Also, in the case of myeloid leukocytes.

In the absence of additives, only a few percent of all leukocytes adhered, but with the addition of differentiation factor pjo-4L.

% adhered within t≠ hours. Approximately 0% of the adherent cells showed phagocytosis via the PC receptor.

Test Example 3 Induction of cytotoxic activity in peripheral blood macrophages A mouse T cell-derived cell line, KL-44, was labeled with isotope 510r and used as a target cell for cytotoxic activity. Human peripheral blood macrophages were pre-treated with differentiation factors, TPA and lymphokine (LK) [human peripheral blood leukocytes were treated with phytoumbilical agglutinin-P (Ditsuyu Co., Ltd. #)]
26-well plate, 1 well per F)! x10' pieces were planted. To these activated macrophages, target cells "0r-1 [fL-4t]/X/0' were added, cultured for 72 hours, and then released into the culture supernatant *"O
The radioactivity of r was measured and the cytotoxic activity was determined by first-order calculation.

*For the maximum free cpm, the value obtained by adding surfactant NP<to(ix) instead of macrophages was used.

Both human peripheral blood-derived macrophages exhibited cytotoxic activity when treated with differentiation factors, similar to those treated with TPA or LK. The results are shown in FIG.

Test example: Hiroshi DMA synthesis inhibitory floating U937 cells! x10'' differentiation factor 1ooryt
After addition of 1H-thymidine (lμC!i)' over time, pulse labeling is performed for 2 hours.

The amount of IH-thymidine uptake by cells (DNA synthesis ability) is 1 after addition of differentiation factor, / without addition of J waiting time, //
/D and becomes macro after 21 hours.

Test Example j Differentiation factor production inducing ability floating U937 cells jXio' cells/go differentiation factor (jl)
U/-) was added and treated for one hour, the medium was replaced with fresh medium, and the differentiation factor newly physiologically secreted into the medium was measured after the cog period, and an activity of about 100 U/d was obtained.

Therefore, it was confirmed that differentiation factor 1 causes proliferating U937 to differentiate and mature, and at the same time causes new differentiation factors to be produced and secreted.

[Brief explanation of the drawing]

FIG. 7 is a diagram showing the cytotoxic activity of TPA (a), the differentiation factor of the present invention (b), and L K (c) on peripheral blood macrophages measured in Test Example 3.

Claims (3)

[Claims] (1) Molecular weight 67,00 derived from human cells capable of differentiating human macrophage progenitor cells into macrophages
A differentiation-inducing factor consisting of 0±5,000 proteins. (2) The differentiation-inducing factor according to claim 1, wherein the human cells are human myeloid leukemia cells. (3) Human myeloid leukemia cells are human monoblast-like cells U9
The differentiation-inducing factor according to claim 2, which is produced by the No. 37 strain.