EXPERIMENTAL AND MOLECULAR PATHOLOGY 19, 320327 (1973)

Studies on the Behavior of Peritoneal Cells Against Soluble Immune Complexes Using the “Agar-Bullet” Method

HISAO YAMAGUCHI, SOHTA NAKA JIMA, HIROSHI TAKEUCHI, CHIKAO TORIKATA, AND KEIZO KAGEYAMA

Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shiniuku-ku, Tokyo, Japan

Received October 4, 1972

The results of our previous investigation indicated that the reaction of intraperi- toneal cells against foreign materials depended on the heterogenicity and the molecu- lar weight of the substance (Takeuchi, 1973). Substances tested for cellular reaction in this agar-bullet method were ( 1) bovine serum albumin, (2) soluble antigen-anti- body complexes, (3) mixtures of bovine serum albumin and rabbit gamma globulin, (4) precipitated complexes, and (5) bovine gamma globulin. The soluble complex prepared with excess antigen in vitro showed a marked cellular reaction, which was considered to be due ‘to its heterogenicity and molecular weight.

It has been demonstrated that soluble antigen-antibody complexes prepared with excess antigen will produce anaphylaxis (Germuth and McKinnon, 1957; Tokuda and Weisner, 1958), smooth muscle contraction (Trapani, Garrey, and Campbell, 1958), lesions of serum sickness (McCluskey et al., 1960) and cu- taneous reactions of increased vascular permeability (Ishizaka and Campbell, 1958; Ishizaka, Ishizaka, and Campbell, 1959).

The initial tissue reactions produced by soluble antigen-antibody complexes are caused mostly by vascular reactions due to increased permeability and emi- gration of inflammatory cells.

Soluble complexes dropped on the normal guinea pig mesentery produced acute local vascular reactions which were characterized by plasma exudation, stasis, sticking, and emigration of leukocytes. Emigrated leukocytes then migrated into perivascular tissue, responding to some chemotactic stimuli.

Although evidences have been obtained that leucocytes respond to antigen- antibody complexes, information on the actual nature of cellular reaction is lacking.

We have devised a new simple experimental method for estimating the cellular reaction to soluble substances (agar-bullet method). Our previous report (Takeuchi, 1973) d emonstrated that the effect of different soluble substances dis- solved in agar on intraperitoneal cells showed a definite cellular reaction, depend- ing on the molecular weight of the substances.

Being based on the fact mentioned above, the study reported here was carried out on intraperitoneal cells to examine the cellular reaction against soluble antigen-antibody complexes prepared in vitro.

Copyri ht 0 1973 by Academic Press, Inc. ,+I ! rigi& 9f reproduction in any form reserved.

SOLUBLE ANTIGEN-ANTIBODY COMPLEX 321

FIG. 1. Apparatus for making agar-bullet, (X) 1.0 ml mess pipet used for making the stick of the bullet; (Y) 2 ml syringe whose top is cut off; (Z) stand.

MATERIALS AND METHODS

Animals. Albino rabbits of both sexes weighing 2.5-3 kg were used. Production of antisera. Rabbits were immunized as follows: 30 mg of bovine

serum albumin (BSA, Armour) in 1 ml of saline was emulsified with an equal volume of complete Freund’s adjuvant. This was injected into foot pads. Two weeks later, intravenous injection of BSA was initiated and administered weekly for 2-6 weeks. The animals were bled one week after the last injection. The anti- BSA sera was pooled and kept frozen at -20°C until used.

Preparation of soluble antigen-antibody complexes. To prepare the soluble complexes, some BSA was added to antisera to reach the point of equivalence. The mixture of antigen and antisera was then refrigerated overnight at 4°C. The precipitates thus formed were washed twice with cold saline. Additional BSA was then added, so that the total amount of BSA reached 20 times the equivalence to enable precipitation, After refrigeration at 4°C overnight with constant stirring, most of the precipitates dissolved in the existing antigen excess. A small amount of undissolved precipitate was removed by centrifugation, and the supernatant fluid was dried in a frozen state.

Preparation of rabbit gamma globulin (RGG). Rabbit gamma globulin was precipitated out of normal rabbit serum by adding saturated ammonium sulphate

agar bullet cut surface

m

microscopic specimen

FIG. 2. Schema of the agar-bullet. (I) Prototype of the bullet; (II) half of the bullet; (III) microscopic specimen.

322 YAMAGUCHI ET AL.

r - -

c

FIG. 3. The cellular reaction cannot be seen in the center stick of the agar-bullets (C) mixed with 5% BSA.

to the serum to give a final concentration of one-third saturation in ammonium sulphate. This was repeated four times, and the precipitate was dialyzed in phos- phate buffered saline. The rabbit gamma globulin fraction prepared by this method was dried in a frozen state.

Technique for estimating celluikr reaction. The procedure for preparing agar is the following: In order to obtain 370 agar saline solution, 1.2 g Bact-agar was dissolved in 40 ml saline solution and kept at room temperature for 12 hr. The mixture was then warmed in a boiled water bath for 15 min until it became a transparent amber color. Then it was cooled to 40°C to be used for the exper- iment.

The procedure for preparing the center sticks of the bullets is the following: Pipets, 0.2 cm diam, were used to suck up the agar solution mixed with protein mater& (5% cont.) in a water bath of 40°C. The pipets were cooled afterward, so that the mixture in them could be used as the center sticks of the bullets.

In order to prepare the outer part of the bullets, 370 agar saline solution was used.

The procedure for making the bullet is the following: The pipet was attached to the supporting apparatus as shown in Fig. 1. The center stick was pushed out of the pipet into the center of a 1.0 cm diam syringe, whose top has been cut off,

and then the 3y0 agar saline solution was carefully poured into the syringe so as not to tilt the center stick of the bullet.

The following protein materials were dissolved in the center stick of the agar-

SOLUBLE ANTIGEN-ANTIBODY COMPLEX 323

FIG. 4. Marked cellular reaction can be seen in the center stick of the agar-bullets (C) mixed with 5% soluble complexes.

bullet to reach 5% in final concentration: (1) BSA, (2) soluble complexes, (3) mixtures of BSA and RGG, (4) precipitated complexes and (5) BGG (Bovine gamma globulin). The agar-bullets inserted into the peritoneal cavity of the rab- bits were taken out after 48 hr. The agar-bullets were then cut vertically through the center of the bullets in half (Fig. 2) and were stained with Hematoxylin and Eosin. The migration of cells into the central stick of the agar-bullets was examined microscopically.

RESULTS

The intraperitoneal cells which migrated into the center stick of the agar- bullets were polymorphonuclear leukocytes and were round or spindle-shaped cells with single nucleus and are thought to have come from peritoneal macro- phage (Fig. 5). Th e same reaction of intraperitoneal cells was obtained from every one of the three rabbits used.

(1) The cellular reaction was not obtained in the agar-bullets in which BSA was dissolved (Fig. 3). Even when the concentration of BSA was increased to as high as 100/o, it elicited no cellular reaction.

324 YAMAGUCHI ET AL.

FIG. 5. Higher magnification of the Fig. 4. The reacting cells are polymorphonuclear leukocytes and round or spindle-shaped cells with single nucleus.

(2) Strong cellular reaction occured in the agar-bullets in which soluble com- plexes were dissolved (Fig. 4). The concentration of anti-BSA gamma globulin contained in the 5% soluble complexes was less than 0.4% of the total protein, The small amount of anti-BSA gamma globulin which conjugated with BSA forming soluble complexes elicited strong stimuli to intraperitoneal cells. The number of the cells found in the agar-bullets mixed with soluble complexes was over twice as many as in those of the bovine gamma globulin, or the mixture of BSA and RGG (20:80).

(3) The intraperitoneal cells did not migrate into the center stick of the agar- bullets in cases where BSA and normal RGG was mixed at the rate of 92.8, 80:20, 50:50. However, a small number of intraperitoneal cells migrated into the agar when the mixture was at the rate of 2O:SO.

(4) No intraperitoneal cells migrated into the center stick of the agar-bullets in which 570 precipitated complexes was mixed.

(5) In the case of heterogenous gamma globulin where 5% BGG was dis- solved in the center stick of the agar-bullets, the intraperitoneal cells migrated into the agar-bullets. This fact was identical with the previous report (Takeuchi, 1973),

DISCUSSION

One of the main dif%culties in studying cellular reaction for soluble substance experimentally has been the lack of suitable techniques. None of the procedures provided satisfactory means of estimating cellular reaction both quantitatively and qualitatively to the soluble substance except the skillful millipore filter

SOLUBLE ANTIGEN-ANTIBODY COMPLEX 325

TABLE I

CELLULAR REACTION IN THE CENTER STICK OF THE BULLET MIXED WITH PROTEIN AND NONPROTEIN MATERIALS

Molecular weignt Cellular reaction

Protein materials

Egg albumin Human albumin Bovine albumin Bovine gamma globulin

Nonprotein substances

PVP PVP PVP PVP Dextran Dextran Dextran Dextran

40,000 70,000 70,000

160,000 +

10,000 -

a~ - 360,000 + 640,000 +

5,006 - 40,000 - 70,000 -

200,000 -I-

chamber devised by Boyden ( 1962). The simple agar-bullet method was designed by us to satisfy the need for such a technique.

In our agar-bullet method, the cells which react to various materials in the agar are not single cells but those of polymorphonuclear leukocytes and are round or spindle-shaped with a single nucleus. The size of the latter cells is also variable (Fig. 5). These cells are generally called intraperitoneal cells and con- sidered to have phagocytic and antibody-forming activity, and are thought to be derived from the peritoneal macrophages. In a wide sense, they form a part of the reticuloendothelial system. The nature of the soluble BSA-anti-BSA complexes responsible for producing increased vascular permeability has been studied by Ishizaka and his associates. Complexes of the Ag,-Ab, molecules and more complicated complexes are capable of producing increased vascular permeability ( Ishizaka, Ishizaka, and Campbell, 1959).

Soluble complexes prepared by us were also considered to have the complexes of Ag,-Ab,. Although RGG, for example, is a large molecular protein, autologous

TABLE II

CELLULAR REACTION IN THE ABAR-BULLETS MIXED WITH PROTEIN MATERIALS

Protein materials (50/O final concn)

BSA Soluble complexes Mixtures of BSA and RGG

BSA/RGG = 92/g BSA/RGG = SO/20 BSA/RGG = 50/50 BSA/RGG = 20/80

Precipitated complexes BGG

Cellular reaction

- +

- - - +

+

326 YAMAGUCHI ET AL.

gamma globulin alone has not elicited cellular reaction in agar-bullet because RGG has no heterogenicity to rabbit. On the other hand, BSA alone, which is a heterogenous protein for rabbit but is a smaller molecule, elicited no cellular re- action in the agar-bullet, as shown in Tables I and II. Even if free BSA not con- jugated with gamma globulin exists in the agar-bullet, and even if a large amount of free BSA goes outside of the bullet, BSA itself does not elicit a cellular reaction, and it is the complex itself made of the large autologous gamma globulin (RGG) and the small heterogenous protein (BSA) which causes the reaction, even if the amount is small. Simple mixture of BSA and RGG show no cellular reaction except when the ratio of BSA and RGG is 20:80, which suggests a mixing of de- generated RGG during the procedures of purification. A large amount of degen- erated autologous gamma globulin is expected to have heterogenic activities, Only the immunologically conjugated protein complex of both kind of proteins can be recognized as the foreign material necessary for the cellular reaction.

It is interesting to know that the soluble immune complex, having the ability to elicit the cellular reaction, is a sort of a large molecular protein complex com- posed of autologous gamma globulin (RGG) and heterogenous (BSA).

Considering the role of the complement for cellular reaction, cellular reaction was observed “even” in the agar-bullet containing nonprotein substance, i.e., a cellular reaction is noted in agar-bullet mixed with large molecular dextran or polyvinyl phosphate (PVP), which are thought to have no immunological rela- tion with complement. Moreover, complement, naturally existing in the peritoneal fluid of rabbits, immerses very quickly into the agar-bullet mixed with BSA or BGG in the early stage after insertion of the agar-bullet into the peritoneal cavity and reacts against both kinds of protein with equal immunological possibilities. However, the cellular reaction is actually observed only in the agar-bullet mixed with BGG, which satisfies the fundamental condition for a cellular reaction- that is, large molecular weight, heterogenous materials. Therefore, the molecular weight of the substances which are contained in agar-bullets play a decisive role concerning cellular invasion into agar-bullet. In our experiments, setting aside heterogenicity, a minimum molecular weight of 100,000 is absolutely necessary for a cellular reaction.

These results lead to the possibility that macromolecularity of the substance is essential for cellular reaction. Accordingly, the soluble complex having the heterogenicity of BSA and forming large molecularity by conjugating BSA with anti-BSA gamma globulin display the strong stimulative effect for cellular reaction.

It has been generally accepted that the protein molecules of larger weight are capable ,of producing antibody more easily than those of smaller weight. This could explain our hypothesis that the reaction of cells for a foreign body includ- ing soluble immune complex depends on the heterogenicity and its molecular weight.

REFERENCES

BOYDEN, S. (1962). The chemotactic effect of mixtures of antibody and antigen on poly- morphonuclear leukocytes. J. Exp. Med. 115, 453-466.

GERMUTH, F. G., and MCKINNON, G. E. ( 1957). Studies on the biological properties of anti- gen-antibody complexes. Bull. Johns Hopkins Hosp. 101, 13-42.

SOLUBLE ANTIGEN-ANTIBODY COMPLEX 327

Isrnz~x~, K., and CAMPBELL, D. H. ( 1958). Biological activity of soluble antigen-antibody complexes. I. Skin reactive properties. Proc. Sot. Exp. Biol. Med. 97, 635-638.

ISHEAKA, K., ISHIZAKA, T., and CAMPBELL, D. H. (1959). Biological activity of soluble anti- gen-antibody complexes. II. Physical properties having skin irritating activity. J. Exp. Med. 109,127-143.

MCCLUSKEY, FL T., BENACERRAF, B., POTIER, J. L., and MILLER, F. (1960). The pathologic effects of intravenously administered soluble antigen-antibody complexes. .I. Exp. Med. 111, 181-194.

TAKEUCHI, H. (1973). The behaviours of the peritoneal macrophages against bovine proteins and other materials of different kinds in molecular weight. ( in press ) .

TOKUDA, S., and WEISER, R. S. (1958). Production of anaphylaxis in the white mouse with soluble antigen-antibody complexes. Science 127, 1237-1238.

TRAPANI, I. L., GARREY, J. S., and CAMPBELL, D. H. (1958). Stimulating action of soluble antigen-antibody complexes on normal guinea pig smooth muscle. Science 127, 700-701.