JP3212428B2 - Method for determining CD18 antigen expression - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for determining the expression of CD18 antigen which is a part of the adhesion protein on the surface of peripheral blood monocytes, and more particularly, to the diagnosis of leukocyte adhesion deficiency based on the presence or absence of CD18 antigen. CD1 to use for the method
8 relates to a method for determining the presence or absence of expression of 8 antigens.

[0002]

2. Description of the Related Art Leukocyte adhesion deficiency (LAD) is one of the immunodeficiencies characterized by severe recurrent bacterial infections seen from birth. . The pathology resembles agranulocytosis, is characterized by delayed umbilical cord shedding in the neonatal period, and the infected wound presents with gangrenous pyoderma disease pathology, such as gingivitis and periodontitis.

[0003] By the way, various cell adhesion molecules are present on the surface of cells such as leukocytes and are involved in important biological reactions such as adhesion between cells, recognition of immune cells, migration of cells, inflammation and the like. I know. However, leukocytes of LAD patients show remarkable adhesion failure, and various leukocyte functions based on cell-cell adhesion are affected, resulting in susceptibility to infection.

[0004] The adhesion molecules include a group of molecules such as the integrin family, the immunoglobulin superfamily, and the selectin family. All of the adhesion molecules belonging to the integrin family are heterodimers consisting of α-chain and β-chain subunits, and the β-chain is β ₂ (CD1
8) is called β ₂ integrin, and LFA-1
(CD11a / CD18), Mac-1 (CD11b / CD18) and p15
0/95 (CD11c / CD18) are known. LA
The leukocytes D patients, the beta ₂ integrin is known to be deficient or extremely reduced.

[0005] By the way, when the target cells are attacked by target cells, such as monocytes, polynuclear leukocytes, K cells or macrophages, the effect cells in charge of cell-mediated immunity bind to the target cells via antibodies on the target cells. And cytotoxic activity (antibody-dependent cellular cytotoxicity: ADCC). That is,
By binding the Fc receptor (FcR) on the effector cell surface to the Fc portion of IgG specifically bound to the target cell surface antigen, the effector cell binds to the target cell to produce a damaging factor or protease. As a result, the target cell is destroyed, whereby the cytotoxic activity is exhibited.

In recent years, as a receptor for human IgG, 3
Different types of Fc receptors have been found. One is FcRI (CD64) expressed on monocytes and macrophages, and the other is expressed on granulocytes, monocytes and macrophages
FcRII (CD32). The third is two different forms of F
cRIII (CD16), one expressed in granulocytes and bound to the cell membrane at the glycosylphosphatidylinositol moiety, the other is natural killer cells (NK cells)
And expressed in macrophages. These Fc
Receptors are known to trigger ADCC. In monocytes, ADCC is released by FcRI and FcRII, respectively.
However, the mechanism of cell lysis by each Fc receptor has not been elucidated.

Conventionally, it was thought that LAD was caused by the deficiency of FcRI and FcRII. However, as described above, it has been found that leukocytes of LAD patients are deficient in β ₂ integrin, which is the cause. It has come to be considered. However, some LADs show typical symptoms and others have a relatively low history of infection.
Although they are called re type and moderate type, the cause of these differences and the pathogenesis of LAD itself are not clear.

On the other hand, it has been shown using herpes simplex virus-infected cells that polynuclear leukocytes of LAD patients do not cause ADCC activity even in the presence of Fc receptor (S. Kohl et al., J. Immunol 137 1688). -16
94 (1986)). This is due to the inability of the effector cells (polynuclear leukocytes) to bind to the target cells (herpes simplex virus infected cells). In contrast, monocytes of LAD patients are known to have ADCC activity.
In addition, ADCC activity of monocytes of healthy subjects is determined by the α of LFA-1.
The addition of monoclonal antibodies against the chains (CD11a) and β-chain (CD18) is markedly inhibited. From these findings,
In multinucleated leukocytes and monocytes, in ADCC activity expression,
An assumption is drawn that the same surface type and the same amount of sticky glycoprotein are not required.

[0009]

SUMMARY OF THE INVENTION As described above, LA
Since the leukocytes of patient D lack the CD18 antigen, a diagnosis can be made by measuring the presence or absence of the expression of the CD18 antigen by flow cytometry. However, a flow cytometer is an expensive device, and is used for diagnosis. Is not economical. Antibodies used for flow cytometry are also expensive and are not universal. Furthermore, in the case of a disease in which an epitope on a cell is blocked,
It has drawbacks such as misdiagnosis.

On the other hand, in the diagnosis of LAD, AD of a mononuclear cell population is used.
Although CC activity measurement has been used to some extent, ADCC expression depends on the type of leukocyte and the mechanism of ADCC is complicated, so it does not lead to a reliable diagnosis, and it is considered that the disease may show other similar disease states. The distinction is difficult, especially when the symptoms are mild. For example, in the above-mentioned measurement of ADCC activity of LAD patients by Kohl et al., No correlation between ADCC activity and LAD was found. In addition, the mechanism of ADCC expression itself is not clear, and the exact role of integrins in ADCC activity expression has not been elucidated.

The present invention has been made in view of the above, and has elucidated the expression mechanism of ADCC, and has further investigated FcRI and FcRII.
Clarifies the mechanism of cell lysis by
An object of the present invention is to provide a method for determining the expression of the CD18 antigen in order to enable diagnosis of D.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that A in peripheral blood monocytes.
DCC is Cc which is mediated by FcRII among Fc receptors.
Those requiring the D18 antigen, but those mediated by FcRI do not require the CD18 antigen.
The present inventors have found that the presence / absence of expression of the D18 antigen can be examined, and have reached the present invention.

[0013] That is, the present invention is characterized in that, among the antibody-dependent cytotoxic activities of peripheral blood monocytes taken from a subject, the antibody-dependent cytotoxic activity expressed through FcRII is measured. This is a method for determining expression. Less than,
The present invention will be described in detail.

<1> Monocyte ADCC and CD18 antigen The expression of CD18 antigen according to the present invention is determined by determining the monocyte A of a patient.
It is performed by measuring DCC activity. For monocytes, Fc
FcRI and FcRII are expressed on the cell surface as receptors,
Although each of these receptors is involved in ADCC expression, among these receptors, ADCC mediated by FcRI (one that requires the intervention of FcRI for binding between target cells and effector cells in ADCC activity expression) is lacking the CD18 antigen. Is also expressed, but FcRI
The present inventors have found that ADCC through I is not expressed without CD18. Therefore, AD through FcRII
By examining the CC activity, the presence or absence of expression of CD18 can be known, and as a result, LAD can be diagnosed.

<2> FcRII-mediated ADCC activity measurement In the present invention, FcRII-mediated ADCC activity was measured by coating the target cell surface with mouse IgG2b (mIgG2b) and suppressing CD18-independent ADCC activity. It is performed by measuring ADCC activity in the state.

FcR of human immunoglobulin G (hIgG)
The binding to I, FcRII, FcRIII is determined by the IgG subclass and affinity, but the four subclasses (hIgG1-
In 4), hIgG1 and hIgG3 bind to all receptors. In contrast, mouse IgG2a (mIgG2a) is known to bind to human FcRI but not to human FcRII, while mouse IgG2b binds to human FcRII but does not bind to human FcRI. This has been demonstrated using the rosette test method (CL Anderson and RJ Looney, 1986 Human
leukocyte IgG Fc receptors. Immunol. Today, 7, 264
-266).

Therefore, by using a cell whose surface is coated with IgG2b as a target cell, FcRII-specific AD
CC activity can be measured. Heretofore, human monocyte ADCC activity test via Fc receptor has been mainly performed using target cells to which rabbit or goat antibodies are bound, but the binding specificity of these rabbit or goat antibodies to human Fc receptor is examined. Has not been reported to be as expensive as mice.

Next, a method for measuring ADCC activity will be described.
Target cells coated with IgG that binds to the surface of erythrocytes and cancer cells are used as target cells, and target cells labeled with RI are incubated with monocytes, which are effector cells, and erythrocytes and cancer cells are destroyed and released. By measuring the radioactivity, ADCC activity can be measured.
For example, sheep red blood cells (SPRC) are coated with trinitrophenylsulfonic acid (TNP), and sheep red blood cells (TNP-SPRC) coated on the surface with TNP are labeled with radioactive chromium, a monoclonal antibody against TNP, and a monocyte. Can be measured by measuring the radioactivity released into the supernatant. At this time, when a mouse IgG2b subclass is used as the anti-TNP antibody, monocyte ADCC induced by FcRII can be selectively measured.

[0019] Since only monocytes express ADCC by this method among mononuclear cells, the ADCC measurement may be performed using a mononuclear cell population. ADC via FcRII
Measuring not only C but also ADCC activity via FcRI at the same time enables more reliable determination of CD18 antigen expression. Further, as a control, the ADCC activity of a healthy person may be simultaneously measured.

[0020]

The following describes the correlation between the expression of ADCC mediated by FcRI and FcRII and the expression of the CD18 antigen.

(1) Preparation of Monocytes Derived from Peripheral Blood of Healthy Subjects and LAD Patients After peripheral blood of healthy subjects or LAD patients is treated with heparin or EDTA and collected, Mono-Poly-resolving Medium (Folw L
aboratories UK) and fractionated into mononuclear cell populations and granulocytes by centrifugation (room temperature, 400 × g, 30 minutes), and the obtained mononuclear cells were cooled in a cold RPMI 1640 medium. After washing 3 times with RPMI 1640 medium containing 10 to 25 mM HEPES, 2 × 10 ⁶ cells /
It was diluted to a concentration of ml and 100 μl was used for the following tests.

(2) Preparation of target cells 0.5 ml of fresh sheep erythrocytes (SRBC, purchased from Denka Seiken)
Is centrifuged (2500 rp) in PBS (phosphate buffered saline).
m, 5 minutes) and suspended in 1 ml of PBS. Next, 1 ml of a 3 mg / ml (PBS) solution of trinitrophenylsulfonic acid (hereinafter referred to as TNP) was added to 1 ml of SRBC, and reacted by shaking at 37 ° C. for 15 minutes, once with PBS and then with gelatin veronal buffer (C
a ² +, containing Mg ² +; hereinafter referred to as GVB). This is called TNP-SRBC. After washing, TNP-SRBC 2 × 10 ⁸ / ml
Was added with 37 mBq of radioactive sodium chromate (Na ₂ ⁵¹ CrO ₄ ) and reacted for 1.5 hours. The obtained radiochromium-labeled TNP-SRBC was washed in the same manner as described above, and suspended in a PBS solution to a concentration of 2 × 10 ⁸ cells / ml.

Next, 0.2 ml of this labeled TNP-SRBC suspension was added to GVB
After adding to 4 ml, anti-TNP-IgG2a monoclonal antibody (mouse: mouse IgG2a subclass for TNP) or anti-TNP-
The IgG2b monoclonal antibody (mouse) was sensitized with a sub-agglutination dose (the last concentration at which erythrocyte agglutination occurs) (37 ° C., 30 minutes), and used as target cells for monocyte ADCC. These antibodies were donated by Assistant Professor Nose of the Department of Pathology, Tohoku University School of Medicine. Below IgG sensitized TNP-SRBC
Is called IgG-TNP-SRBC. IgG-TNP-SRBC was adjusted to 2 × 10 ⁷ cells / ml after washing with GVB.

(3) Method of measuring ADCC activity To each well of a round-bottom 96-well microtiter plate, 1 × 10 ^{4 to} 5 × 10 ⁶ cells / ml of effector cells and 100 μl of target cells were added at 37 ° C. _The cells were cultured in _two incubators. At this time, the target cells were ⁵¹ Cr-TNP-SRBC and 10μ
1 anti-TNP-IgG2a antibody or ⁵¹ Cr-TNP-SRBC and 10 μl of anti-TNP-IgG2b may be added. Twenty hours later, 100 μl of the centrifuged supernatant of the reaction solution in each well was collected, and the radioactivity was measured using a gamma counter. The measurement result was expressed as a% ⁵¹ Cr release according to Equation 1, and this was defined as ADCC activity.

[0025]

[Formula 1]% ⁵¹ Cr release = (C ₁ / C ₀ ) × 100 where C ₁ is the radioactivity (cpm) of the centrifuged supernatant, and C ₀ is released when the target cells are treated with 10% sodium dodecyl sulfate. Radioactivity (cpm)

(4) Results of ADCC Measurement of Peripheral Blood Monocytes of Healthy Subjects ADCC activity of peripheral blood mononuclear cells of healthy subjects is determined by mIgG2a-sensitized TNP-
15% when SRBC is used as labeled cells, mIgG2b-sensitized TNP-SR
It was 16% when sensitized with BC. At this time, the ratio (E / T ratio) between the effector cells and the target cells was 1:10.

The mononuclear cell population was subjected to the adhesion method using the wells of a microtiter plate, and ADCC of adhered cells and non-adhered cells was measured in the same manner.
Mononuclear cells (2 × 10 ⁵ cells), adsorbed cells (0.7 × 10 ⁵ cells) or non-adsorbed cells (1.3 × 10 ⁵ cells) and ⁵¹ Cr-labeled TNP-SRBC
And (2 × 10 ^6), incubated the anti-TNP-IgG2a or anti-TNP-IgG2b presence, ADCC activity was measured. Table 1 shows the results.

[0028]

[Table 1]

As apparent from the results, ADCC activity was observed only in the adherent cells. The mononuclear cell population includes lymphocytes and monocytes, and among these, the only adherent cells are monocytes.
CC activity.

(5) LAD patients (2) Results of ADCC measurement of peripheral blood mononuclear cells Next, LAD patients (,) and healthy subjects (,,)
MIgG2a-sensitized TNP-SRBC or
ADCC when mIgG2b-sensitized TNP-SRBC is used for target cells
Activity was measured. The results are shown in Table 2 for patients and Table 3 for patients.

[0031]

[Table 2]

[0032]

[Table 3]

As shown in the table, when measuring ADCC activity, the ratio of effector cells to target cells was 1:10, 1: 3,
When the measurement was performed while changing the ratio to 1: 1, when mIgG2a-sensitized TNP-SRBC was used as the target cell, the ADCC activity increased in a concentration-dependent manner in a control healthy person. ADCC activity also increased in LAD patients, but was lower than in healthy subjects.

On the other hand, when mIgG2b-sensitized TNP-SRBC was used as the target cells, ADCC was observed in healthy subjects as the E / T ratio changed.
Although an increase in activity was observed, little activity was observed in patients.

The results indicate that monocytes of the LAD patient were FcRI, Fc
Since it is known to have both RII antigen, mIg
Even if FcRII to which G2b specifically binds is present, ADCC activity is not expressed without the CD18 antigen. On the other hand, it is suggested that the Fc receptor to which mIgG2a specifically binds, that is, ADCC mediated by FcRI, is expressed even without the CD18 antigen.

(6) Inhibition Experiment of Peripheral Blood Monocyte ADCC in Healthy Individuals Using Integrin Antibodies ADCC activity of normal human peripheral blood mononuclear cells was measured by using various concentrations of anti-CD18 monoclonal antibody F (ab ') ₂ fragment ( The antibody was measured in the presence of a hybridoma-derived antibody MAY-017 antibody produced by the Institute for Research on Mycobacteriosis of Tohoku University. For target cells, mIgG2a-sensitized TNP-SRBC and mIgG2b-sensitized
The measurement was performed at an E / T ratio of 1/10 using TNP-SRBC. Table 4 shows the results. In the following, ADCC activity when mIgG2a-sensitized TNP-SRBC was used as a target cell was referred to as FcRI-ADCC, mIgG2b.
ADCC activity using sensitized TNP-SRBC as target cells
Described as RII-ADCC.

[0037]

[Table 4] From this result, when CD18 is blocked with an anti-CD18 antibody, Fc
It can be seen that the RI-ADCC activity is only slightly reduced, while the FcRII-ADCC activity is significantly suppressed. Therefore, expression of FcRII-ADCC is not CD18.
Is required, and the above-mentioned finding that FcRI-ADCC is expressed even in the absence of CD18 is supported.

As described above, among the monocyte ADCC activities, those mediated by FcRII are not expressed without the presence of the CD18 antigen.
Can be examined. Since the leukocytes of LAD patients do not express or are extremely low on the CD18 antigen,
Measuring the FcRII-ADCC activity enables diagnosis of LAD.

In addition, expression of FcRII-ADCC requires CD18
It is considered that the above-mentioned finding that FcRI-ADCC is expressed even in the absence of CD18 can be used for elucidation of the ADCC activity expression mechanism and the like.

[0040]

Embodiments of the present invention will be described below. In each of the following examples, monocyte ADCC activity was measured in the same manner as described above.

[0041]

EXAMPLE 1 First, an example of determining CD18 antigen expression by ADCC activity of monocytes taken out from a LAD patient will be described together with a diagnosis example using the results.

For LAD patients and healthy subjects, monocyte AD
CC activity was measured at an E / T ratio of 1/10. The results are shown in the upper part of Table 5. In addition, the results of the same measurement of ADCC activity on the same patient and other healthy subjects on the same day are shown in the lower part of Table 5.

[0043]

[Table 5] From this result, a patient whose FcRII-ADCC was significantly lower than that of a healthy subject was determined to not express the CD18 antigen, and as a result, this patient was diagnosed as having LAD.
In addition, this patient separately purchased a monoclonal antibody (DAKO MHM2
3 (anti-CD18) and Becton-Dickinson's Leu15 (anti-CD11b)) were used to examine the expression of CD11 / CD18, confirming that it was LAD.

[0044]

Embodiment 2 Next, an example in which LAD is suspected but not LAD using the method of the present invention will be described.

[0045] CD18 antigen expression was determined by the method of the present invention for patients who had repeated infections suspected of having LAD. Table 6 shows the results.

[0046]

[Table 6] This patient had no reduction in FcRII-ADCC.
Deficiency was found, and she was diagnosed with chronic granulomatosis. This patient was diagnosed with chronic granulomatosis early on, and
Treatment with a T combination (trimethoprim + sulfamethoxazole) was started.

[0047]

Embodiment 3 Next, a description will be given of a diagnosis example in which the method of the present invention has been used to diagnose a non-LAD.

For other patients suspected of having LAD, the expression of the CD18 antigen was determined by the method of the present invention. As a result, as shown in Table 7, since FcRII-ADCC did not decrease, it was determined that the CD18 antigen was expressed, and a detailed examination was performed. As a result, cancer was found in the esophagus. The patient surgically removed the cancer.

[0049]

[Table 7]

[0050]

According to the present invention, monocyte ADCC contains CD18.
It was revealed that there are FcRII-mediated ones that require the antigen and FcRI-mediated ones that do not require the CD18 antigen,
It has become possible to determine the presence or absence of expression of the D18 antigen. By utilizing this, a reliable diagnosis of LAD can be made.

Claims (3)

(57) [Claims] 1. A CD characterized by measuring an antibody-dependent cytotoxic activity expressed through FcRII among antibody-dependent cytotoxic activities of peripheral blood monocytes taken out from a subject.
Method for determining 18 antigen expression. 2. The method according to claim 1, wherein the antibody-dependent cytotoxicity expressed through FcRII is measured by measuring peripheral blood monocytes taken from a subject and sheep erythrocytes coated with trinitrophenylsulfonic acid. And anti-trinitrophenylsulfonic acid mouse IgG2b antibody in the solution,
A method for determining CD18 antigen expression, which is performed by quantifying sheep red blood cell components released into a solution. 3. The method according to claim 1, wherein the antibody-dependent cytotoxicity expressed through FcRI is further measured.
A method for determining CD18 antigen expression, which is compared with an antibody-dependent cytotoxic activity expressed through I.