실험적 급성 혈청병성 사구체신염에 대한 prednisolone 및 cyclophosphamide의 영향

Abstract

[한글]

The Influence of Prednisolone and Cyclophosphamide on the Experimental Acute Serum

Sickness Glomerulonephritis

Yong Bae Lee, M.D.

Department of Medical Science The Graduate School Yonsei University

(Directed by Professors: Yoo Bock Lee, M.D. and In Joon Choi, M.D.)

In a majority of the cases, the initial glomerular insult appears to be triggered

by immunologic processes. As a result of experimental studies in the past decade it

appears that these various experimental models of human glomerulonephritis fit into

two general immunopathologic mechanisms each dependent upon the action of

circulating antibodies(Dixon, 1968; Germuth, 1973). The first of these mechanisms,

which is known as anti-glomerular basement membrane disease, involves antibodies

capable of reacting with antigens in or of the glomeruli, usually the glomerular

basement membrane(GBM) The second depends upon the formation of circulating

antibody-non-glomerular antigen complexes, some of which become trapped in the

glomeruli. Either of these two immunopathologic mechanisms of antigen-antibody

reaction in the glomerular tufts initiate an inflammatory process. The second

mechanism is immunologically nonspecific. There is no immunologic relationship

between the antibody in the circulating complexes and the glomeruli. In the human

being, more than 95% of glomerulonephritis of immunologic origin is due to

circulating antigen-antibody complexes. The most reliable method of differentiating

these two forms of glomerulonephritis is to observe the distribution of

immunoglobulins (Ig) and complement (C') in the glomeruli by immunofluorescence.

The first suggestion that circulating immune complexes might cause nephritis came

from studies of acute, or "one-shot",serum sickness. The apparent coexistance of

heterologous serum protein antigen and antibody in the circulation, plus the rapid

disappearance of antigen just prior to the appearance of antibody, led to the

conclusion that antigen and antibody in the circulation form a "toxic compound"

responsible for serum sickness (Von Pirquet, 1911). The original and detailed

studies of antigenic clearance in the acute serum sickness-induced animals have

been done (Germuth, 1953: Dixon, 1962). In our country, experimental studies of the

circulating immune complex mechanism, particularly, about secondary immune reaction

and anaphylaxis, were done by Yun and his collagues (1928 and 1956).

Steroids are known to suppress undesirable immune or inflammatory responses.

Although there exists many descriptive reports dealing with these steroids,

relatively little is known about the exact mechanisms (Zurier and Weissmann, 1973).

The value of corticosteroids in the treatment of adult patients with the nephrotic

syndrome is small because this syndrome is largely confined to those with mild or

minimal lesions of the glomeruli. For this reason, for the treatment of focal

glomerulosclerosis and the some of the adult glomerulonephritis with

corticosteroid-resistant diseases, the use of cytotoxic and antimetabolite drugs

alone or in combination with corticosteroids appear effective(White, et al., 1966 :

Sharptstone, et al. 1969:Bardana, Jr., et al., 1970).

It seems interesting and helpful to whether or not the mechanisms involved

steroid and cyclophosphamide inhibition of glomerular damage in acute serum

sickness are due to a marked inhibitory effect on the cellular exudation, on the

penetration of immune complexes into the wall of the glomerular loop, or to

interruption of translocation of the preformed circulating immune complexes. In

addition, it seems interesting to study the mesangial function of immune deposits

in relation to mesangium.

The present study, therefore, is designed to compare the effect of prednisolone,

given in conventional dosage, with that of cyclophosphamide, particularly about the

immune deposits in the glomerular tufts for anti-inflammatory and anti-immunologic

effects, by light, immunofluorescent and electron microscopic studies.

Materials and Methods

A. Materials:

A total of twenty eight albino male rabbits weighing approximately 2 Kg. each

were used. For the induction of "one shot" acute serum sickness, crystalized Bovine

Serum Albumin from the Miles Laboratory was used. A total of 10 Gm. of crystalized

Bovine Serum Albumin contained in 100 ml. Phosphate buffer saline after autoclaving

was processed by membrane filtration. A filter membrane (Schleicher and Schnell,

0.45 micron) was put on the Filter holder(S and s, 27 mm.), which was connected to

filtering flask, and processed in an autoclave for 15 minutes at 15 1bs. Renal

biopsy specimens obtained with the Vim-Silverman needle (14 gauge), sections of

kidney from unilateral nephrectomy and from postmortem examinations were frozen in

dry ice-acetone mixture, for immunofluorescent examination.

A high-titered rabbit anti-BSA serum was used for the daily capillary

precipitation reactions. Prednisolone from the Dong Sin Laboratory and

cyclophosphamide from the Philip laboratory were used for the anti-inflammatory and

anti-immunologic effects.

B. Experimental Methods:

The animals for the experiment were divided into three groups: group Ⅰ was

Normal Control, 5 rabbits: group Ⅱ was Experimental group, 18 rabbits; and group

Ⅲ was phosphate buffer saline control group, 5 rabbits. Group Ⅱ was subdivided

into A, B, and C. The subgroup A was an experimental control, 6 rabbits; B was the

prednisolone-treated group, 6 rabbits: C was the cyclophosphamide-treated group, 6

rabbits. These eighteen rabbits of experimental group Ⅱ were injected

intravenously with 0.5 Gm. crystalized BSA contained in 5 ml. phosphate buffered

saline. Beginning on the fourth day and continuing daily thereafter, the 12 rabbits

of subgroup B and C were treated intramuscularly with 1.5mg. per Kg. of

prednisolone for subgroup B, and with 2.5 mg. per Kg, of cyclophosphamide for

subgroup C. Clearance of BSA from the circulation was checked in all rabbits by

daily capillary precipitation reactions using high-titered rabbit anti-BSA

serum(Germuth, F.G., Jr., et al.,1957). On the day of complete or maximum BSA

Clearance, a renal biopsy and unilateral nephrectomy were performed, and later all

animals were sacrificed on the twenty-first and second experimental day. Kidney

tissues for immunofluorescent examination were cut in three to four micron

thickness, and were examined by a direct immunofluorescent antibody technique with

specific fluorescein-tagged antisera fractions, such as FITC-conjugated anti-rabbit

immunoglobulins, (IgG, IgM, IgA), anti-rabbit beta 1 C globulin and rabbit anti-HSA

serum. All of the above reagents, crystalized Bovine Serum Albumin, rabbit anti-BSA

serum , and FITC-conjugated anti-rabbit fractions were supplied by Dr. Germuth

Experimental Pathology Laboratory.

The kidney tissues for histopathologic examination were fixed in 10% formalin and

stained with hematoxylin and eosin, and also with periodic acid Schiff stain.

Ultrastructural examination was done with usual processing and double staining

methods, and done by Hitachi HU-11E Electron microscope.

Discussion & Summary

The correlative studies by light, immunofluorescent and electron miscroscopic

examinations of the effects of prednisolone and cyclophosphamide on the renal

glomerular lesions induced by experimental acute serum sickness was made and

following results are obtained.

1. The severity of the glomerulonephritis appeared greater under light microscopy

of specimen from rabbits having a rapid antigenic clearance. The speed of antigenic

clearance was delayed in the prednisolone-treated group in comparing with either

the experimental control or the cyclophosphamide-treated groups.

2. Fellowing the clearance of antigen from the circulation, immunoglobulin,

complement (C'3), and BSA (antigen) were deposited in a finely granular form with

the distribution along the glomerular capillary loop in the experimental control

group, but mainly within the mesangium both in the prednisolone-treated and

cyclophosphamide-treated groups. These deposits appeared more prominent in the

cyclophosphamide-treated group than in the prednisolone-treated group. These

findings confirm that circulating immune complexes are localized on the glomerular

tufts and cause inflammatory changes.

3. By immunofluorescent and electron microscopy, the circulating antigen-antibody

immune complexes were localized in the subepithelial portion and intramembranous

area in the experimental control group, but were localized in the subendothelial

portion and mesangium in both the prednisolone-treated and cyclophosphamide-treated

groups. In some of these, however, subepithelial and intramembranous deposits were

rarely, or only occasionally, found.

4. By electron microscopy of these prednisolone-treated and

cyclophosphamide-treated groups, transient or partial mesangial interposition,

sometimes. were impressively found, particularly in the sacrificed rabbits.

These data indicate that prednisolone and cyclophosphamide inhibit the

development of immunologic glomerular lesions. However, there appears to be a

different inhibitory mechanism for these two drugs because a delayed antigenic

clearance was noted particularly in the prednisolone-treated group. The present

experiment adds further support to the previously reported hypothesis that serum

sickness glomeroulonephritis results from the transmigration of circulating soluble

immune complexes across the wall of the glomerular loop. The anti-inflammatory and

anti-immunologic effects of prednisolone and cyclophosphamide, appear to induce

molecular reorientation and aggregation of immune complexes within the

subendothelial layer. Also these cause interruption of transmigration of

macromolecular antigen-antibody complexes across the glomerular loop wall, so that

glomerular injury and inflammation do not develop. These immune complexes of

specific macromolecule within the subendothelial zone of the glomerular loop appear

to contact mesangial cells by transient or partial mesangial interposition. Hence

they are available to localize in the mesangium, where, it may be assumed, they may

be phagocytozed or passed toward the hilus of the glomerulus by way of potential

intercellular canaliculi. However, it is net possible to exclude the possibility

following the use of these two drugs, that some of the molecular reoriented

specific macromolecules of immune aggregates may first accumulate in the mesangium

of the glomeruli.

[영문]

In a majority of the cases, the initial glomerular insult appears to be triggered by immunologic processes. As a result of experimental studies in the past decade it appears that these various experimental models of human glomerulonephritis fit into

two general immunopathologic mechanisms each dependent upon the action of circulating antibodies(Dixon, 1968; Germuth, 1973). The first of these mechanisms, which is known as anti-glomerular basement membrane disease, involves antibodies capable of reacting with antigens in or of the glomeruli, usually the glomerular basement membrane(GBM) The second depends upon the formation of circulating antibody-non-glomerular antigen complexes, some of which become trapped in the glomeruli. Either of these two immunopathologic mechanisms of antigen-antibody

reaction in the glomerular tufts initiate an inflammatory process. The second mechanism is immunologically nonspecific. There is no immunologic relationship between the antibody in the circulating complexes and the glomeruli. In the human being, more than 95% of glomerulonephritis of immunologic origin is due to

circulating antigen-antibody complexes. The most reliable method of differentiating these two forms of glomerulonephritis is to observe the distribution of immunoglobulins (Ig) and complement (C') in the glomeruli by immunofluorescence. The first suggestion that circulating immune complexes might cause nephritis came

from studies of acute, or "one-shot",serum sickness. The apparent coexistance of heterologous serum protein antigen and antibody in the circulation, plus the rapid disappearance of antigen just prior to the appearance of antibody, led to the conclusion that antigen and antibody in the circulation form a "toxic compound"

responsible for serum sickness (Von Pirquet, 1911). The original and detailed studies of antigenic clearance in the acute serum sickness-induced animals have been done (Germuth, 1953: Dixon, 1962). In our country, experimental studies of the circulating immune complex mechanism, particularly, about secondary immune reaction and anaphylaxis, were done by Yun and his collagues (1928 and 1956).

Steroids are known to suppress undesirable immune or inflammatory responses. Although there exists many descriptive reports dealing with these steroids, relatively little is known about the exact mechanisms (Zurier and Weissmann, 1973). The value of corticosteroids in the treatment of adult patients with the nephrotic syndrome is small because this syndrome is largely confined to those with mild or minimal lesions of the glomeruli. For this reason, for the treatment of focal glomerulosclerosis and the some of the adult glomerulonephritis with corticosteroid-resistant diseases, the use of cytotoxic and antimetabolite drugs

alone or in combination with corticosteroids appear effective(White, et al., 1966 : Sharptstone, et al. 1969:Bardana, Jr., et al., 1970).

It seems interesting and helpful to whether or not the mechanisms involved steroid and cyclophosphamide inhibition of glomerular damage in acute serum sickness are due to a marked inhibitory effect on the cellular exudation, on the penetration of immune complexes into the wall of the glomerular loop, or to

interruption of translocation of the preformed circulating immune complexes. In addition, it seems interesting to study the mesangial function of immune deposits in relation to mesangium.

The present study, therefore, is designed to compare the effect of prednisolone, given in conventional dosage, with that of cyclophosphamide, particularly about the immune deposits in the glomerular tufts for anti-inflammatory and anti-immunologic effects, by light, immunofluorescent and electron microscopic studies.

Materials and Methods

A. Materials:

A total of twenty eight albino male rabbits weighing approximately 2 Kg. each were used. For the induction of "one shot" acute serum sickness, crystalized Bovine Serum Albumin from the Miles Laboratory was used. A total of 10 Gm. of crystalized

Bovine Serum Albumin contained in 100 ml. Phosphate buffer saline after autoclaving was processed by membrane filtration. A filter membrane (Schleicher and Schnell, 0.45 micron) was put on the Filter holder(S and s, 27 mm.), which was connected to filtering flask, and processed in an autoclave for 15 minutes at 15 1bs. Renal biopsy specimens obtained with the Vim-Silverman needle (14 gauge), sections of kidney from unilateral nephrectomy and from postmortem examinations were frozen in dry ice-acetone mixture, for immunofluorescent examination.

A high-titered rabbit anti-BSA serum was used for the daily capillary precipitation reactions. Prednisolone from the Dong Sin Laboratory and cyclophosphamide from the Philip laboratory were used for the anti-inflammatory and anti-immunologic effects.

B. Experimental Methods:

The animals for the experiment were divided into three groups: group Ⅰ was Normal Control, 5 rabbits: group Ⅱ was Experimental group, 18 rabbits; and group Ⅲ was phosphate buffer saline control group, 5 rabbits. Group Ⅱ was subdivided into A, B, and C. The subgroup A was an experimental control, 6 rabbits; B was the prednisolone-treated group, 6 rabbits: C was the cyclophosphamide-treated group, 6 rabbits. These eighteen rabbits of experimental group Ⅱ were injected intravenously with 0.5 Gm. crystalized BSA contained in 5 ml. phosphate buffered saline. Beginning on the fourth day and continuing daily thereafter, the 12 rabbits of subgroup B and C were treated intramuscularly with 1.5mg. per Kg. of prednisolone for subgroup B, and with 2.5 mg. per Kg, of cyclophosphamide for subgroup C. Clearance of BSA from the circulation was checked in all rabbits by daily capillary precipitation reactions using high-titered rabbit anti-BSA serum(Germuth, F.G., Jr., et al.,1957). On the day of complete or maximum BSA Clearance, a renal biopsy and unilateral nephrectomy were performed, and later all animals were sacrificed on the twenty-first and second experimental day. Kidney tissues for immunofluorescent examination were cut in three to four micron

thickness, and were examined by a direct immunofluorescent antibody technique with specific fluorescein-tagged antisera fractions, such as FITC-conjugated anti-rabbit immunoglobulins, (IgG, IgM, IgA), anti-rabbit beta 1 C globulin and rabbit anti-HSA serum. All of the above reagents, crystalized Bovine Serum Albumin, rabbit anti-BSA serum , and FITC-conjugated anti-rabbit fractions were supplied by Dr. Germuth Experimental Pathology Laboratory.

The kidney tissues for histopathologic examination were fixed in 10% formalin and stained with hematoxylin and eosin, and also with periodic acid Schiff stain.

Ultrastructural examination was done with usual processing and double staining methods, and done by Hitachi HU-11E Electron microscope.

Discussion & Summary

The correlative studies by light, immunofluorescent and electron miscroscopic examinations of the effects of prednisolone and cyclophosphamide on the renal glomerular lesions induced by experimental acute serum sickness was made and following results are obtained.

1. The severity of the glomerulonephritis appeared greater under light microscopy of specimen from rabbits having a rapid antigenic clearance. The speed of antigenic clearance was delayed in the prednisolone-treated group in comparing with either

the experimental control or the cyclophosphamide-treated groups.

2. Fellowing the clearance of antigen from the circulation, immunoglobulin, complement (C'3), and BSA (antigen) were deposited in a finely granular form with the distribution along the glomerular capillary loop in the experimental control group, but mainly within the mesangium both in the prednisolone-treated and cyclophosphamide-treated groups. These deposits appeared more prominent in the cyclophosphamide-treated group than in the prednisolone-treated group. These findings confirm that circulating immune complexes are localized on the glomerular

tufts and cause inflammatory changes.

3. By immunofluorescent and electron microscopy, the circulating antigen-antibody immune complexes were localized in the subepithelial portion and intramembranous area in the experimental control group, but were localized in the subendothelial portion and mesangium in both the prednisolone-treated and cyclophosphamide-treated groups. In some of these, however, subepithelial and intramembranous deposits were rarely, or only occasionally, found.

4. By electron microscopy of these prednisolone-treated and

cyclophosphamide-treated groups, transient or partial mesangial interposition, sometimes. were impressively found, particularly in the sacrificed rabbits.

These data indicate that prednisolone and cyclophosphamide inhibit the development of immunologic glomerular lesions. However, there appears to be a different inhibitory mechanism for these two drugs because a delayed antigenic clearance was noted particularly in the prednisolone-treated group. The present

experiment adds further support to the previously reported hypothesis that serum sickness glomeroulonephritis results from the transmigration of circulating soluble immune complexes across the wall of the glomerular loop. The anti-inflammatory and anti-immunologic effects of prednisolone and cyclophosphamide, appear to induce molecular reorientation and aggregation of immune complexes within the subendothelial layer. Also these cause interruption of transmigration of macromolecular antigen-antibody complexes across the glomerular loop wall, so that glomerular injury and inflammation do not develop. These immune complexes of

specific macromolecule within the subendothelial zone of the glomerular loop appear to contact mesangial cells by transient or partial mesangial interposition. Hence they are available to localize in the mesangium, where, it may be assumed, they may

be phagocytozed or passed toward the hilus of the glomerulus by way of potential intercellular canaliculi. However, it is net possible to exclude the possibility following the use of these two drugs, that some of the molecular reoriented specific macromolecules of immune aggregates may first accumulate in the mesangium of the glomeruli.