JPH10227792A - Measuring method for neutrophil chemotactic factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure neutrophil migration activity of neutrophil chemotactic factor, by using peritoneal exudate neutrophil of stimulus inoculated animals, and quantifying the neutrophil which migrationally fell down from an upper room of a migrational chamber to a lower room containing a chemotactic factor in colorimetric determination method using a hemocyte measuring device (MTT). SOLUTION: Gradually diluted chemotactic factors are put on the lower chamber divided with the migrational membrane using 2 wells per dilution, and objects which have no chemotactic factor, and fMLPs as a positive control are put on it by two wells respectively. In the test of neutrophil migration, not only culturing place of animal cells but also saline solution are used. The neutrophil of rabbits and others gathered from a peritoneum for measuring migration capacity is cleaned with saline solution and used. Cell buoyant liquid which is adjusted to specific concentration is put into the upper room, and is migrated in a carbon dioxide incubator at prescribed temperature for 30-60min., and the number of cells which migrationally fell down to the lower chamber is counted. The migrating cells are measured using colorimetric determination method with a colter counter or a measuring device for number of hemocytes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring a neutrophil chemotactic factor, which comprises using neutrophils exuded from the peritoneal cavity of an animal inoculated with a stimulant.

[0002]

2. Description of the Related Art It is well known that infiltration of leukocytes into tissues plays an important role in the body defense mechanism. In host defense reactions against local infections, neutrophils accumulate at infected inflammatory sites to kill foreign bacteria and the like.

[0003] In a biological defense reaction against cancer cells or virus-infected cells, leukocytes are mobilized and accumulated at a target local area. In these biological defense reactions, the action of recruiting and accumulating leukocytes (neutrophils) to inflammation sites and target sites, that is, the action of inducing leukocyte migration and chemotaxis, plays an important role.

Conventionally, neutrophil chemotaxis factors include:
Complement fragment C5a (J. Biol. Chem., 2536955-6964,
1978), Leukotriene B4 (Prostaglandines, 20, 411)
-418,1980), human monocyte-derived neutrophil chemotactic factor (Proc. Nat
l.Acad.Sci.USA, 84,9233-9237,1987), fMLP (folmyl
Methyonyl Loycyl Phenylalanine), PAF (Platelet activ
ating factor) is known, but recently, IL-1 (Interleukin-
1) and TNF (Tumor necrosis factor), LPS (Lipopolysacch
a new neutrophil chemotactic factor, IL-8 (Interleukin-8) and its family, which are synthesized and secreted by monocytes, fibroblasts, endothelial cells, etc. (Cell Engineering, 9,435-443, 1990).

[0005] IL-8 has been found to be a neutrophil chemotactic factor and a neutrophil activator that causes degranulation. It also has chemotactic activity on basophils, eosinophils, and vascular endothelial cells, and various new biological activities due to neutrophil activation have been reported. For example, release of intracellular enzymes, promoting the production of LTB _4, Candida albicans growth inhibition, enhanced expression of cell adhesion factors in neutrophils (Mac-1, CR-1 ), unstimulated endothelial cells to neutrophils Effects on non-hemocyte cells such as enhanced adhesion, decreased adhesion of endothelial cells to IL-8-stimulated neutrophils, and stimulation of skin keratinocyte proliferation and accumulation of melanoma cells at the administration site are also reported. It became so.
However, among these biological activities, neutrophil migration activity is the strongest activity as a direct effect.

[0006] In recent years, neutrophil chemotactic factors have been measured using highly sensitive enzyme-labeled immunoassays (ELISA) and radioimmunoassays, which have been produced using a specific monoclonal antibody production technology that has been rapidly developed in recent years. However, these do not reflect biological activity, and it has been desired to establish a simple method for measuring biological activity with excellent accuracy and reproducibility.

[0007] The biological activity measurement methods reported so far for human IL-8 include a method for measuring neutrophil migration activity, a lysozyme enzyme and active oxygen released by neutrophil activation. There are methods. Currently used migration activity measurement methods have problems in sensitivity, accuracy, reproducibility, and simplicity in operation as described below. Methods for measuring the release of lysozyme enzyme and the release of active oxygen are not available. , IL-8, which is an indirect effect of neutrophil activation, and thus has problems in sensitivity, specificity, and reproducibility.

[0008] The migration activity of chemotactic factors is measured by the Boyden method (J.
Exp.Med., 115, 453-466, 1962).
munology 10,409-416,1966, Am.J.Pathol.64,521-530,
1971, J. Immunol.Methods 46, 251-258, 1981, J. Clin. P
athol 35, 182-185, 1982) have been studied and used individually. However, these methods are not standardized,
There are drawbacks such as the fact that the measured value is easily influenced by the measurer, lacks objectivity and has a problem with accuracy, and requires a great deal of labor to count stained migrating cells under a microscope. It was impossible to process.

Recently, Gallin et al. (J. Immunol., 110, 2)
33-240, 1973) and Kurihara et al. (J. Pharm. Dyn., 7, 747-754, 19
84) has previously been a problem with Cr-labeled neutrophils
We devised a chemotaxis assay that could cover the shortcomings of the Boyden method, but this method also did not allow for the screening of large numbers of samples, required special research equipment, and the dangers of using radioisotopes. Isotope labeling has problems such as a decrease in neutrophil viability and its function.

In addition, Watanabe et al. (Japan. J. Pharmacol., 3
9,102-104,1985, J.Pharmacol.Methods, 22,13-18,198
9) devised a multi-well type Boyden chamber and developed a system that can measure neutrophil chemotaxis of many samples using this. Although this method has the advantage of being relatively quick and convenient, it is suitable for screening assays, but requires a skilled technique to fix the membrane that separates the cell suspension from the upper and lower chambers from the sample solution, and requires a high cell count. Since it is measured from the turbidity absorbance, there remains a problem in terms of accuracy compared to the measurement method using a Coulter counter.

On the other hand, neutrophils activated by IL-8 cause degranulation, releasing enzymes such as elastase, cathepsin G, myeloperoxidase, β-glucuronidase, collagenase, and type IV gelatinase, and release active oxygen. It is known to release. The method of measuring the released lysozyme enzyme cannot be detected unless the cells are destroyed with Tryton X-100 or the like, and high reproducibility and high precision measurement cannot be expected. In addition, since the neutrophil activating effect of IL-8 is very weak and the release of active oxygen alone is small, a combination with a strong activating factor such as TNF or GM-CSF is required to measure the amount of active oxygen. It is necessary to provide a measurement system with excellent accuracy, sensitivity, and reproducibility (Blood, Vol. 78, No.
10,2708-2714,1991).

There are several established methods for preparing neutrophils. Usually, a method often used is to fractionate and purify from human or animal blood using a specific gravity separation solution. This is done by mixing blood collected from a vein or heart using a heparinized syringe with dextran saline and leaving it at room temperature for 30 minutes to 1 hour to separate the plasma layers (polynuclear leukocytes, lymphocytes, monocytes, This is a method of purifying neutrophils (including platelets) using a specific gravity separation solution adjusted to an appropriate specific gravity. In the migration test using the neutrophil fraction thus obtained, several problems have been pointed out. Even if elaborate purification is repeated, contamination of erythrocytes and the like is inevitable, and it takes time and effort to obtain a high-purity neutrophil fraction, resulting in lower neutrophil yield, blood sampling The long time and procedure from purification to purification tend to cause a decrease in neutrophil function, affecting the reproducibility of migratory ability measurement. Contamination of erythrocytes etc. affects the migration test. It is difficult and there are large individual differences between humans and animals. In addition, when human blood is used, there is a risk of infection and difficulty in regular supply.

As a simpler method for separating neutrophils,
There is a method in which glycogen or casein is inoculated into the abdominal cavity of an animal and neutrophils exuded into the abdominal cavity are used. In this method, the concentration and time of the stimulant to be inoculated vary depending on the animal species, but neutrophils with a purity of 95% or more can be obtained under appropriate conditions, and large amounts of neutrophils can be easily collected. There are advantages that can be done. However, the function of this method may be slightly different from that of normal neutrophils by using a stimulant. Numerous advantages result if they can be used to measure the migratory activity of neutrophil chemotactic factors.

There is also a method for obtaining neutrophils from bone marrow. This is a method in which bone marrow fluid is collected from the femur of an animal using a syringe, and a neutrophil fraction is obtained using the above-described specific gravity separation solution in the same manner as in purification from blood. This method has the same long drawbacks as the blood fractionation, and is too time-consuming to use routinely. In addition, the yield and function of neutrophils may differ from experiment to experiment, so it is not appropriate. Absent.

Usually, the neutrophil migration activity of a neutrophil chemotactic factor is measured by using a neutrophil obtained by blood fractionation to count the number of migrating cells on a chemotaxis membrane under a microscope. I have. In this method, cells that migrated to the back of the membrane must be stained by the double staining method. Since errors due to uneven staining are likely to occur, and the cells do not migrate uniformly on the membrane, microscopy is required. There are many problems in terms of accuracy and reproducibility, such as less objectivity in the number of cells measured below. Inoculating the animal with the stimulant in the abdominal cavity,
If a migration test can be performed using a large amount of neutrophils exuding into the abdominal cavity and the number of migrated cells can be measured using a device such as a Coulter counter, an objective measurement of migration activity can be performed more easily. Also, if the reactivity of stimulant-activated peritoneal exudate neutrophils to chemotactic factors is enhanced compared to normal peripheral blood neutrophils, excellent migration activity measurement that can support the measurement of large samples A law could be built.

[0016]

As described above, the conventional methods for measuring migration ability have a problem, and there is no standardized method that can evaluate a large number of samples with high accuracy. It is desired to establish an activity measurement method.

[0017]

The above-mentioned object is achieved by the present invention described below. In other words, as a result of intensive studies based on the above-mentioned problems, we succeeded in simply measuring the neutrophil migration activity of neutrophil chemotactic factors with good accuracy and reproducibility,
The present invention has been completed.

That is, the present invention is a method for measuring neutrophil migration activity of neutrophil chemotactic factor, comprising the following method. Using neutrophils exuded from the peritoneal cavity of the animal inoculated with the stimulant, neutrophils that had migrated and dropped from the upper chamber of the migration chamber to the lower chamber containing the chemotactic factor were analyzed using a Coulter counter, a blood cell counter, an absorbance analyzer, Neutrophil chemotaxis factor determined by colorimetry using a tetrazolium salt ((3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, hereinafter referred to as MTT)) Is a method for measuring neutrophil migration activity.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION As animals used in the present invention, rabbits, guinea pigs, rats or hamsters are used, but are not particularly limited thereto.
These animals weigh 1-3 kg for rabbits and 0,0 for guinea pigs.
2-1kg, rat 150-250g and hamster 80-150g
Is preferably used.

Neutrophils of these animals can be prepared by the following method. Collection of peritoneal exudate neutrophils requires inoculation of the peritoneal cavity with a stimulant. The concentration of stimulants and the time at which neutrophil infiltration peaks vary among animal species, and must be harvested under conditions where 90% or more of the exuded cells are neutrophils. Glycogen, casein, polypeptone, thioglycolate medium and the like are used as stimulants. Preferably, shellfish glycogen (Sigma) or Oyster glycogen (Sigma) is used after autoclaving. In rabbits, glycogen is dissolved using physiological saline, and in shellfish glycogen, the final concentration is
Prepare and inoculate at a concentration of 0.15% and 0.1% for oyster glycogen. For animals other than rabbits, dissolve in distilled water and use as a 10% concentration solution.

The stimulant is intraperitoneally inoculated with 150 ml for rabbits, 10 ml-20 ml for guinea pigs, and 5 ml and 2 ml for rats (about 200 g) and mice (about 20-25 g), respectively. The peak of infiltration of polynuclear leukocytes into the peritoneal cavity is around 12-16 hours in guinea pig rabbits and around 6 hours in mice and rats after stimulant inoculation. Care must be taken because as time elapses after stimulation, monocytes and macrophages increase and the purity of neutrophils decreases.

There is no significant difference between males and females in the degree of neutrophil exudation and migration ability. In addition, there is no apparent difference in the migratory function of neutrophils obtained from animals of different ages, strains, and breeding environments, but preferably 5-10
Week-old SPF (specific pathogen free) or clean bred animals are used.

The advantage of using neutrophils exuded from the peritoneal cavity after inoculation of the stimulant is evident from the comparison with the following method using neutrophils obtained by fractionating and purifying from whole blood or peripheral blood as a control. It is.

To separate neutrophils from the blood of humans and various animals, for example, an immunological experiment procedure (Nankodo, 635)
Page, 1995), but the method is not particularly limited thereto. Human peripheral blood is collected from the arm vein, and blood from animals such as rabbits, guinea pigs, rats and mice is collected from the heart using a disposable syringe moistened with heparin. Multinuclear leukocytes are fractionated from the collected blood, for example, as follows.
Heparinized blood was diluted 2-fold with Hanks' solution, and 1% of this heparinized blood was mixed with 1 / 4-1 / 5 volume of 3% dextran saline, mixed well, and then mixed at room temperature for 30 minutes. Upon standing, it is separated into a plasma layer containing polynuclear leukocytes, lymphocytes, monocytes, and platelets, and a red blood cell layer. Separate the upper plasma layer,
Ficoll-Hypaque solution (Pharmacia) or Ficoll-Conra
Gently overlay the solution (IBL) and centrifuge at 400 g for 30 minutes at room temperature. Neutrophils are obtained as a sediment, and the supernatant is removed. Erythrocytes mixed into the sediment are subjected to hypotonic treatment with distilled water for injection to lyse hemolysate, returned to isotonic with PBS, and then 280 g for 10 min.
Collect neutrophils by centrifugation at 0 ° C. Wash this 2-3 times with Hanks' solution and use it as a neutrophil fraction for the migration test.

In the neutrophil migration test of the present invention, the migration cell (chemotaxel, Kurabo, etc.) is placed on a multiwell type Boyden chamber or a 24-well plate in which the upper and lower chambers are separated by a polycarbonate filter. Although a method may be used, a method using individually separated migration cells is preferred from the viewpoint of the recovery rate of the migration cells. As the migration membrane, one having a pore size of 3 to 8 μm is used, and a 3 μm membrane is preferably used.

Next, the actual method of measuring the migration activity of neutrophil chemotactic factors will be described. The serially diluted chemotactic factor was placed in the lower chamber partitioned by the migration membrane using 2 wells per dilution, and 10 ^-7 M fMLP was added to each of the control containing no chemotactic factor and the positive control for 2 times.
Put on each well. The liquid volume of the sample to be put in the lower chamber is an amount that the membrane that separates the upper and lower chambers is immersed.

In the neutrophil migration test, a saline solution such as PBS or Hanks solution can be used in addition to a usual culture medium for animal cells. In addition, 0.1% (w / v) bovine serum albumin, 10-20 mM HEPES,
Penicillin G (0.1 mg / ml) and the like can also be contained. However, in each case, 1-
Addition of ₂ mM CaCl ₂ is essential.

Rabbit and guinea pig neutrophils collected from the abdominal cavity for migratory activity measurement are passed through a mesh to remove dust and tissue fragments mixed at the time of collection, and washed 3-4 times with a migration medium or a salt solution. Then use for measurement. 2 in upper room
Add the cell suspension prepared at a concentration of x10 ⁶ cells / ml, and add
In a carbon dioxide gas incubator (about 5% CO ₂ concentration)
Let go for 30 to 60 minutes. Maximum number of migrating cells is 30-40
A plateau is reached in minutes, and no increase in the number of migrating cells is observed even after a longer time. When the migration time is over, the migration membrane or the migration cells are removed, and the number of cells that have migrated and dropped into the lower chamber is counted.

In order to efficiently collect neutrophils that have migrated to the lower chamber, it is necessary to minimize the adhesion of neutrophils to the chamber or plate. When a 24-well plate is used, a plate made of polystyrene is preferably coated with collagen or gelatin, if possible, since neutrophils are strongly adhered to the cells and it takes a long time to recover the cells.
The migrated cells recovered from the lower chamber are analyzed by a Coulter counter, a blood cell counter, an absorbance measurement, and a colorimetric method using a tetrazolium salt (MTT method) (J. Immunol. Method. 65,
55-63, 1983).

According to the present invention, for example, the neutrophil migration activity of human IL-8 is equal to or higher than that of human neutrophils using guinea pig peritoneal exudate neutrophils (maximum migration activity concentration: 6-12 ng / m). The present invention can be applied to the measurement of all neutrophil chemotactic factors other than human IL-8. For example, human IL-8 subfamily, rabbit IL-8 / NAP-1, rat GRO / CINC, mouse MIP-2, bovine IL-8, complement fragment C5a, leukotriene B4, fMLP, etc. can be measured according to the present invention. It is not limited to these.

The standard required for measuring neutrophil migration activity is as follows:
A purified product with high purity is used. This sample is an ELISA
Method, amino acid composition analysis, absorbance measurement, etc.
The amount of IL-8 protein is measured, aliquoted so that it can be used each time the migration ability is measured, and stored in a freezer at -80 ° C. The unit of the human IL-8 measurement standard is corrected to the titer of the WHO international standard (1000 units / ampoule) before use.

The neutrophil migration activity of the neutrophil chemotactic factor according to the present invention is analyzed as follows. (A) Subtract the value of the negative control (the number of cells in the medium or buffer alone containing no neutrophil chemotactic factor) from each measured value obtained as the number of migrated cells. (B) Calculate the ratio of the measured values showing the maximum migratory activity as 100 and the value of the negative control as 0 in each concentration of the sample which is two-fold serially diluted with n = 2. (C) The abscissa plots the concentration of the chemotactic factor in logarithm, and the ordinate plots the ratio to the concentration showing the maximum migration activity obtained in (b). From (d) plots performs regression positive correlation is obtained, the ratio from the regression equation number of migrated cells determine the concentration equivalent to 50, the ED ₅₀ value. (E) titers are determined as the reciprocal of the dilution that gives the ED ₅₀ values. (F) The value obtained in (e) is corrected with the value of the laboratory standard that has been reconciled with the titer of the WHO international standard product.

[0033]

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples.

Example 1 Preparation of peritoneal exudate neutrophils of various animals: Oyster glycogen (Sigma, G-8751) solution used as a stimulant was prepared by dissolving 0.2 g of rabbit solution in 200 ml of physiological saline and adding 150 m
l was inoculated intraperitoneally. For guinea pigs, rats and mice, prepare a 10% solution with distilled water for injection.
-12 ml, 5 ml for rats, and 2 ml for mice. Rabbits were fixed with 2.0-2.5 kg of New Zealand White females, and inoculated intraperitoneally with 50 ml of the stimulant in three 50 ml portions using a 50 ml syringe. Guinea pigs are 7 weeks old Hart
Lay females were inoculated with 10 ml of stimulant, and in both cases, peritoneal exudate neutrophils were collected 16 hours later. Rats and mice are
Six hours after inoculation with the stimulant, peritoneal exudate was collected. Peritoneal exudate was 0.02% heparin (Novo Nordisk A / S, 10
(00 units / ml), suspended in PBS, centrifuged, washed twice more, centrifuged (1000 rpm, 5 minutes), and then passed through a 70-um mesh to remove solids such as tissue fragments. . The cells collected by centrifugation were washed twice with PBS supplemented with 0.1% BSA and 1 mM CaCl ₂ and centrifuged (1000 rpm, 5 minutes) to prepare neutrophils used in the experiment.

Both rabbits and guinea pigs can be
1-5 × 10 ⁸ cells were obtained per animal, and 1-3 × 10 ⁷ cells were obtained in rats and mice. To clarify the types of cells obtained, cells collected from the guinea pig peritoneum were placed on cytospin (SHANDON, Cytospin 3), centrifuged at 650 rpm for 5 minutes, and the filter paper was dried. Cells were stained with the staining solution to identify the type of cells. The results confirmed that 90% or more of these cells were neutrophils.

Example 2 Purification of neutrophil fractionation from human and animal blood: Human blood was collected from the arm vein with a 50 ml syringe moistened with heparin (40 ml), and dispensed in 20 ml portions into two 50 ml centrifuge tubes. PBS in centrifuge tube
(Hanks solution) Add 20 ml and 3 ml of 10% dextran solution, and add 30 ml.
Let stand at room temperature for minutes. The supernatant containing neutrophils was collected, layered in equal volumes on a centrifuge tube containing Ficoll-Hypaque solution (Pharmacia) adjusted to a specific gravity of 1.077, and centrifuged at room temperature at 1700 rpm for 30 minutes. Neutrophils are obtained as sediment, so remove the supernatant, add an appropriate amount of distilled water to lyse the contaminating erythrocytes, pipette twice, and immediately add PBS containing 0.1% BSA and 1 mM CaCl ₂ to PBS. Add 5 volumes and centrifuge (100
(0 rpm, 5 minutes) and the supernatant was removed. Neutrophils obtained as a precipitate were suspended in the above PBS, and the number of cells was counted using trypan blue, and used for a migration test. 3 x 10 from 40 ml of human blood
⁷ 1 x 10 ⁸ neutrophils were obtained.

Rabbit and guinea pig blood was collected entirely from the heart. Rabbits used 2.5 kg, New Zealand white breeds, and guinea pigs used females of all 22-week-old Hartlay breeds. Neutrophil fractionation was performed in the same manner as described above for fractionation from human peripheral blood. As a result, it was observed that neutrophils accounted for more than 90%.

Example 3 Fractionation and Purification of Rat Bone Marrow Neutrophils: 8-week-old Wistar (13
0-150g) and SD (170-190g) rats
Bone marrow fluid was collected from the femur and the neutrophils contained therein were fractionated. PBS with 2.5% fetal bovine serum in the femoral cavity
Was flushed several times with a 5 ml syringe equipped with a 23G injection needle, and bone marrow fluid was collected. The number of cells obtained at this time was about 2-5 × 10 ⁷ per femur. This was overlaid on a 1.077 specific gravity separation solution in an equal volume and centrifuged at 1800 rp for 25 minutes at room temperature. Erythrocytes mixed in the neutrophil fraction precipitated at the bottom of the centrifuge tube were hemolyzed in the same manner as in Example 2, and centrifuged again to purify neutrophils. Neutrophils of about 70% purity were obtained by this method.

Example 4 Migration test: The migration test was carried out by placing chemotaxel (Kurabo) having a hole of 3 μm on a 24-well collagen-coated plate. The sample was serially diluted two-fold, and 500 ul was added to each well of a 24-well plate, on which chemotaxel was placed. Migration buffer (PBS, 0.1% BS) in the cell
A, 1mMCaCl ₂ ) with neutrophil suspension adjusted to 2 x 10 ⁶ / ml.
200 ul was inoculated and allowed to migrate in a 5% CO2 incubator (37 ° C) for 40-60 minutes. The migration time was kept constant every experiment. After the migration time, the chemotaxel was gently removed from each well of the 24-well plate, and the cells migrated and dropped into the wells were measured using a Coulter counter.

Example 5 Comparison of reactivity of peritoneal exudate neutrophils of various animals to human IL-8: Guinea pig (Hartley, 10 weeks old), mouse (C3H, 7 weeks old), rat (SD, 7 weeks old) ) And rabbits (New Zealand White, 12 weeks old) were used to prepare neutrophils according to the method described in Example 1 using females. Using these cells, a migration test was performed according to the method described in Example 4. As shown in FIG. 1, guinea pigs and rabbit neutrophils show a migration response to 0.1-100 ng / ml of human IL-8, whereas mouse and rat cells migrate to these concentrations of IL-8. Did not. Above all, guinea pig peritoneal exudate neutrophils showed strong reactivity with human IL-8.

Example 6 Comparison of reactivity between neutrophils derived from guinea pig heart blood and neutrophils exuding peritoneal cavity to human IL-8: Three out of four female guinea pigs of Hartley type, 22 weeks old, were used. Whole blood was collected from the heart, and neutrophils were separately prepared according to the method described in Example 2. The other one was inoculated with 10% oyster glycogen, and neutrophils were prepared according to the method described in Example 1. Using these neutrophil fractions, the migration test shown in Example 4 was performed to examine the migration ability of neutrophils. 2 and 3 show the results. Neutrophils fractionated from the blood of the heart were obtained by multiplying the maximum number of cells migrated by IL-8 to the number of cells migrated to a buffer containing no human IL-8 (chemotaxis).
It can be seen that the reactivity of neutrophils exuded from the abdominal cavity is 50 times or more, while the index is about 5 times at the maximum.

Example 7 Comparison of the reactivity of neutrophils derived from rabbit heart blood and neutrophils exuded to the peritoneal cavity to human IL-8: 2.5 kg of New Zealand White female rabbits using the method described in Example 2 According to the above, whole blood was collected from the heart and neutrophils were fractionated. On the other hand, peritoneal exudate neutrophils were obtained by the method shown in Example 1 using another rabbit. From the whole blood, 2 x 10 ⁷ neutrophils were fractionated from one animal, and about 2 x 10 ⁸ neutrophils were exuded from the peritoneal cavity. Using these neutrophils, the migration test shown in Example 4 was performed, and the migration ability was compared. The results are shown in FIGS. 4 and 5, where neutrophils obtained by fractionation from whole blood were
It did not show any migration reaction to L-8. On the other hand, in the peritoneal exudate neutrophils, the ratio of the maximum number of migrated cells by IL-8 to the number of non-specific migrated cells to the buffer containing no IL-8 was about 10 times. It was also confirmed in rabbits that peritoneal exudate neutrophils showed high reactivity to human IL-8.

Example 8 Comparison of reactivity between rat bone marrow neutrophils and peritoneal exudate neutrophils to human IL-8: Rat bone marrow neutrophils were used for each of 8 female SD and Wister rats. It was prepared from the femur according to the method described in Example 3 to obtain 1-1.5 × 10 ⁷ neutrophils. Peritoneal exudate neutrophils were collected 6 hours after intraperitoneal inoculation with 5 ml of 10% oyster glycogen. A migration test was performed using these neutrophils, and the results shown in FIGS. FIGS. 6 and 7 show the results of examining the migration activity of human IL-8 on bone marrow neutrophils using rat IL-8 (CINC) as a control. The sensitivity was low, indicating that the maximum concentration of migratory activity was required to be at least 50 times that of guinea pig peritoneal exudate neutrophils. 8 and 9 show higher concentrations of human IL.
The results of examining the reactivity of peritoneal exudate neutrophils using -8 showed that both Wister and SD rats had a maximum migration response equivalent to that of guinea pigs at 5 ug / ml. This is 500 times the maximum migration concentration for neutrophils. These results indicate that human IL-8 has a remarkably low migration activity on rat neutrophils irrespective of the origin of neutrophils and the species of rat.

Example 9 Comparison of migration reaction between human peripheral blood neutrophils and guinea pig peritoneal exudate neutrophils against human IL-8: Human peripheral blood neutrophils were fractionated and purified according to the method described in Example 2. In this way, 2-4 × 10 ⁷ neutrophils were obtained from 40 ml of blood. Guinea pigs were 9-week-old female Hartley breeds. Peritoneal exudate was collected 16 hours after inoculation with oyster glycogen, and neutrophils were obtained according to Example 1. The migration test of these neutrophils was performed as described in Example 4, and the reactivity to human IL-8 was compared. The results in FIG. 10 were obtained by examining the migration ability of neutrophils fractionated from different human blood. FIG. 11 compares the migration ability of neutrophils obtained from another person with guinea pig peritoneal exudate neutrophils. From these results, human I
It was found that the reactivity of human peripheral blood neutrophils to L-8 varied greatly between individuals, and that the sensitivity of guinea pig peritoneal exudate neutrophils was equal to or higher than that of human peripheral blood neutrophils.

Example 10 Reactivity of Guinea Pig Peritoneal Exudate Neutrophils to Various Human Chemokines: The reactivity of guinea pig peritoneal exudate neutrophils to various human chemokines was investigated and is shown in FIG. This revealed that guinea pig peritoneal exudate neutrophils also reacted in a concentration-dependent manner with other human chemokines having neutrophil migration activity other than IL-8.

Example 11 Comparison with the Boyden Chamber Method in Measuring the Number of Migrating Cells: The method for measuring the number of migrating cells used in the present invention using guinea pig peritoneal exudate neutrophils having high sensitivity to human IL-8 was compared with the conventional method. Was compared with a method of counting the number of cells that had migrated and adhered to the Chemotaxis membrane. The Boyden chamber used was 48 wells, and the membrane was a polycarbonate filter (Neuro Prob) with 3 um holes.
e Inc., 25 x 80 mm). The lower chamber was filled with 30 ul of a three-fold serially diluted human IL-8 and a buffer-only control, and the upper chamber was filled with 50 ul of a guinea pig neutrophil solution adjusted to 2 × 10 ⁶ / ml in each well. This chamber
The cells were placed in a 5% carbon dioxide incubator and allowed to migrate for 40 minutes at 37 ° C. After the migration time, remove the membrane filter, scrape the cells in the upper chamber, fix the migrated cells with alcohol, stain with Diffick stain (International reagent), air-dry, and count the migrated cells under a microscope (x400). did. The results are shown in FIG. 13. Although it was possible to determine the maximum migration activity concentration by this method, it was difficult to draw a clear concentration-dependent curve. On the other hand, in the method shown in Example 4, as shown in FIGS.
It has been found that it is possible to draw a concentration-dependent curve excellent in linearity even for −8.

Example 12 Examination of the migration time giving the maximum migration activity: The migration time in the migration test shown in Example 4 using guinea pig peritoneal exudate neutrophils was divided into 30, 60 and 90 minutes, respectively, to determine the number of migrated cells. Was examined for changes. The results are shown in FIG. From this, it was found that neutrophil migration reached a plateau almost 30 minutes after the start of migration, and that the number of migrated cells hardly changed after 90 minutes. Example 13 Daily reproducibility of migratory activity: Using a migration test method using guinea pig peritoneal exudate neutrophils according to the present invention, the protein amount was determined to be 1 ug / ml by ELISA, absorbance measurement, and amino acid composition analysis. Table 1 shows the results of measuring the migration activity of human IL-8 on different measurement dates. The activity unit is the reciprocal of the dilution factor of the IL-8 concentration (ED ₅₀ ) that gives 50% of the number of migrated cells, with the average of the measured values showing the maximum migration activity as 100% and the value of the buffer-only negative control as 0. Is shown as the number of units. As shown in FIG. 18, the abscissa represents the logarithm of IL-
The maximum number of migrating cells (subtracting the number of negative control cells) was taken as 100 on the vertical axis, and the ratio of the number of migrating cells at the IL-8 concentration of 4 to 5 points with good linearity was calculated. After plotting, a regression equation was obtained and the ED ₅₀ value was calculated.

[0048]

[Table 1] Example 14 Correlation between migratory activity and ELISA method: Results of 37 migration tests using guinea pig peritoneal exudate neutrophils according to the present invention
The correlation with the result measured by the ELISA method was examined and shown in FIG. The abscissa plots the logarithm of the IL-8 concentration measured by the ELISA method, and the ordinate plots the logarithm of the activity unit obtained in the migration test. It can be seen that there is an excellent correlation between the two.

Example 15 Influence of Neutrophil Drawing Processing on Neutrophil Migration Ability: Example 6
Peritoneal exudate neutrophils and peripheral neutrophils IL-
In order to clarify that the difference in the reactivity to neutrophils was not due to the operation of fractionating neutrophils from blood, peritoneal exudate neutrophils were treated in the same manner as in the fractionation of peripheral blood, and migratory ability was evaluated. Examined. Four 9-week-old guinea pigs were prepared, and neutrophils were prepared from the three guinea pigs according to Example 2. The remaining one is from Example 1.
Neutrophils were collected according to the procedure described above, and the same operation as that of the peripheral blood component of Example 2 was performed for half of the neutrophils (2 × 10 ⁸ ). Using these neutrophils, a migration test on human IL-8 was performed,
The result is shown in FIG. From this, it was confirmed that the difference in reactivity between neutrophils exuding abdominal cavity and neutrophils in the peripheral blood was not due to the difference in neutrophil adjustment operation at night.

[0050]

Industrial Applicability According to the present invention, the migration activity of neutrophil chemotactic factor can be easily measured with high accuracy and reproducibility.

[Brief description of the drawings]

FIG. 1 shows the results of examining the migration response of peritoneal exudate neutrophils of various animals to human IL-8.

FIG. 2 shows the results of the migration reaction of guinea pig peritoneal exudate neutrophils to human IL-8.

FIG. 3 shows the results of chemotactic response of guinea pig cardiac neutrophils to human IL-8.

FIG. 4 shows the results of a migration reaction of rabbit peritoneal exudate neutrophils to human IL-8.

FIG. 5 shows the results of a migration reaction of rabbit heart blood neutrophils to human IL-8.

FIG. 6 shows the results of a migration reaction of Wister rat bone marrow neutrophils to human IL-8.

FIG. 7 shows the results of a migration reaction of SD rat bone marrow neutrophils to human IL-8.

FIG. 8 shows the results of the migration reaction of Wister rat peritoneal exudate neutrophils to human IL-8.

FIG. 9 shows the results of a migration reaction of SD rat peritoneal exudate neutrophils to human IL-8.

FIG. 10 shows the results of a migration reaction of human peripheral blood neutrophils to human IL-8.

FIG. 11 shows the results of a comparison of the migration reaction between guinea pig peritoneal exudate neutrophils and human peripheral blood neutrophils against human IL-8.

FIG. 12 shows the results of examining the reactivity of guinea pig peritoneal exudate neutrophils to various human chemokines.

FIG. 13 shows the results of a migration reaction of guinea pig peritoneal exudate neutrophils to human IL-8 in a conventional method using a Boyden chamber.

FIG. 14 shows the concentration dependence of human IL-8 on migratory activity of guinea pig peritoneal exudate neutrophils (log plot of 0.01-50 ng / ml).

FIG. 15 shows the concentration dependency of human IL-8 on the migration activity of guinea pig peritoneal exudate neutrophils (0-0.8 ng / ml).

FIG. 16 shows the concentration dependency of human IL-8 on the migration activity of guinea pig peritoneal exudate neutrophils (0 to 12.5 ng / ml).

FIG. 17 shows the results of examining the migration time that gives the maximum migration activity.

FIG. 18. Human IL using guinea pig peritoneal exudate neutrophils
The correlation between the measured value of the migration activity of -8 and the concentration of human IL-8 is shown.

FIG. 19: Human IL using guinea pig peritoneal exudate neutrophils
The correlation between the measured value of the migration activity of -8 and the value measured by the ELISA method is shown.

FIG. 20 shows that the migratory function of guinea pig peritoneal exudate neutrophils is higher than that of peripheral blood fractionated neutrophils, not due to the effect of fractionation treatment.

Claims (8)

[Claims] 1. A method for measuring a neutrophil chemotactic factor, comprising using neutrophils exuded from the peritoneal cavity of an animal inoculated with a stimulant. 2. The method according to claim 1, wherein the stimulant is a glycogen, casein, polypeptone or thioglycolate medium. 3. The method for measuring a neutrophil chemotactic factor according to claim 1, wherein the animal is a guinea pig, rabbit, rat or hamster. 4. The method for measuring a neutrophil chemotactic factor according to claim 1, wherein the neutrophil chemotactic factor is derived from a human. 5. The method according to claim 1, wherein the neutrophil chemotactic factors are the interleukin 8 subfamily (CXC chemokine) and the MCAF subfamily (CC chemokine). 6. The neutrophil chemotactic factor is interleukin 8,
The method for measuring a neutrophil chemotactic factor according to claim 1, which is fMLP, leukotriene B4, activated complement fifth component (C5a) and TNF-α. 7. The neutrophil chemotactic factor according to claim 1, wherein neutrophils that have migrated and fallen from the upper chamber of the migration chamber to the lower chamber containing the neutrophil chemotactic factor are counted. Migration activity measurement method. 8. The neutrophil chemotactic factor according to claim 1, wherein the neutrophil count is counted by a colorimetric method using a Coulter counter, a blood cell count measuring device, an absorbance measuring device, and a tetrazolium salt. Measuring method.