Journal oflmmunological Methods, 26 (1979) 315--323
315
© Elsevier/North-Holland Biomedical Press
ATTACHMENT OF POLYMORPHONUCLEAR LEUKOCYTES TO GLOMERULI WITH IMMUNE DEPOSITS *
T. YAMAMOTO, I. KIHARA, T. MORITA and T. OITE
Department of Pathology, Institute of Nephrology, Niigata University School of Medicine, Asahimachidori 1, Niigata 951, Japan (Received 10 September 1978, accepted 1 October 1978)
A method for quantitative evaluation of a biological activity of immune complexes deposited in glomeruli is described. The activity reflects the activation of complement and is represented by the number of PMN attached to a glomerulus. It is possible to compare data from different individuals or different phases of glomerulonephritis. The complement c o m p o n e n t concerned is considered to be C3, activated through the classical or alternate pathway.
INTRODUCTION
Immunofluorescent studies on renal biopsies have provided significant advances as they have afforded an insight into the pathogenesis of glomerulonephritis (Mellors and Ortega, 1956; McCluskey, 1971; Wilson and Dixon, 1974). Although immune complexes detected with immunofluorescent technique are widely considered to play a role in tissue injury (Dixon et al., 1961; Germuth and Rodriguez, 1973), it is not satisfactorily proved whether the deposited immune complexes induce tissue damage and whether they are biologically active in causing inflammation. From this point of view, a complement fixation test has been applied to sections of tissue to demonstrate the avidity of immune complexes for complement (Burkholder, 1961; Kozima and Vogt, 1970). With this method, however, the activation of complement could not be quantitatively estimated. The purpose of this report is to demonstrate that some immune complexes deposited in glomeruli can attract polymorphonuclear leukocytes (PMN) in vitro and such biological activity of immune deposits is quantitated by counting PMN adherent to glomeruli.
* Supported partially by Research Grant for Specific Disease (Chronic Nephritis) from the Ministry of Health and Welfare and a Grant-in-aid for Scientific Research 148140 of Ministry of Education.
316 MATERIALS AND METHODS
Sections
Specimens of kidney were obtained from rats with glomerulonephritis induced by bovine serum albumin (BSA) as previously described (Yamamoto et al., 1978). They were snap-frozen in liquid n-hexane at --70°C. Sections were made with a Minotome (Damon/IEC Division) by setting the scale at 1 and placed in a bath of cold 0.01 M phosphate-buffered saline, pH 7.2, (PBS) and air-dried. To evaluate the effect of conditions, specimens from rats with moderate or marked proliferative glomerulonephritis were used. Fresh rat s e r u m
Wistar rats were anesthetized with ether. Blood was collected by cutting the axillary vessels, allowed to clot at room temperature for 10 min and centrifuged at 4°C. When needed, serum was diluted with isotonic Veronalbuffered saline, pH 7.6 containing 0.15 mM CaC12 and 0.5 mM MgC12 (VB). Heat-inactivated serum
Fresh rat serum was inactivated at 56 ° C for various periods of time. Anti-C3 treatment of serum
Antiserum for the third c o m p o n e n t of complement (C3) was prepared by the modified m e t h o d of Mardiney and Miiller-Eberhard (1965). A crude globulin fraction of anti-C3 was obtained by precipitation of the sera in 33% saturated a m m o n i u m sulfate at 4°C and dialysed against VB. The globulin fraction was adjusted by dilution with VB to a concentration of 38 mg/ml. As a control, normal rabbit sera were treated with the same procedure as anti-C3 sera. One volume of several dilutions with VB of anti-C3 globulin fraction or normal globulin fraction was mixed with 4 vol of fresh rat serum diluted 1/10 with VB and placed at 4°C for 48 h and then the precipitate was spun down. PMN were suspended in the supernatant and incubated with sections. PMN
PMN were obtained from the peritoneal cavity of Wistar rats by the technique of Hirsch and Church (1960). Ten milliliters of 0.1% glycogen was injected intraperitoneally and the exudate was collected 3 h later and centrifuged at 800 rev/min for 10 min. The cell pellet containing over 95% PMN was resuspended in serum or diluted serum to various densities. Incubation
The equipment used is shown in Fig. 1. A glass slide with 6 sections was covered with another glass slide with two ribbons of electric insulating tape stretched across the slide. The distance between the two glass slides was
317
electrical insulating tape I
glass~slide
,± 0.2 mm I.
JT
glass slide with sections PMN suspended in serum Fig. 1. The e q u i p m e n t used for i n c u b a t i o n .
approximately 0.2 mm. PMN suspension was discharged into the space with a Pasteur pipet and the equipment was placed at 37°C in'a moist atmosphere for 40 min in the standard assay and for various periods of time (10--60 min) to examine the effect of incubation time. Then, the slide with sections was placed in a bath of cold PBS, rinsed gently with PBS, fixed with ethanol and stained with hematoxylin and eosin. Assessmen t PMN on 60 glomeruli of moderate size were counted and the results represented the average number of PMN on a glomerulus. Immunofluorescent technique As reported previously (Yamamoto et al., 1978), the sections were stained with fluorescein isothiocyanate-labeled anti-BSA, anti-rat IgG, and anti-rat C3. The intensity and the a m o u n t of fluorescence were arbitrarily graded as --, +, ++, and +++. RESULTS
Standard procedure When PMN were suspended in fresh rat serum to a concentration of 2 × 107/ml and incubated with sections of rat BSA nephritis, PMN were observed microscopically on glomeruli with i m m u n e deposits and n o t other regions o f the sections. As seen in Fig. 2a and b, PMN attached to glomeruli were countable with ease and the calculation of PMN on a certain glomerulus in serial sections did n o t give much difference. Precise location of PMN was u n k n o w n , but suspected to be closely associated with immune complexes as defined by the immunofluorescent technique. Application to a group o f rat BSA-nephritis As revealed in Table 1, about a hundred PMN were attached to a severely
O
•
•
,
....
:
315
© Elsevier/North-Holland Biomedical Press
ATTACHMENT OF POLYMORPHONUCLEAR LEUKOCYTES TO GLOMERULI WITH IMMUNE DEPOSITS *
T. YAMAMOTO, I. KIHARA, T. MORITA and T. OITE
Department of Pathology, Institute of Nephrology, Niigata University School of Medicine, Asahimachidori 1, Niigata 951, Japan (Received 10 September 1978, accepted 1 October 1978)
A method for quantitative evaluation of a biological activity of immune complexes deposited in glomeruli is described. The activity reflects the activation of complement and is represented by the number of PMN attached to a glomerulus. It is possible to compare data from different individuals or different phases of glomerulonephritis. The complement c o m p o n e n t concerned is considered to be C3, activated through the classical or alternate pathway.
INTRODUCTION
Immunofluorescent studies on renal biopsies have provided significant advances as they have afforded an insight into the pathogenesis of glomerulonephritis (Mellors and Ortega, 1956; McCluskey, 1971; Wilson and Dixon, 1974). Although immune complexes detected with immunofluorescent technique are widely considered to play a role in tissue injury (Dixon et al., 1961; Germuth and Rodriguez, 1973), it is not satisfactorily proved whether the deposited immune complexes induce tissue damage and whether they are biologically active in causing inflammation. From this point of view, a complement fixation test has been applied to sections of tissue to demonstrate the avidity of immune complexes for complement (Burkholder, 1961; Kozima and Vogt, 1970). With this method, however, the activation of complement could not be quantitatively estimated. The purpose of this report is to demonstrate that some immune complexes deposited in glomeruli can attract polymorphonuclear leukocytes (PMN) in vitro and such biological activity of immune deposits is quantitated by counting PMN adherent to glomeruli.
* Supported partially by Research Grant for Specific Disease (Chronic Nephritis) from the Ministry of Health and Welfare and a Grant-in-aid for Scientific Research 148140 of Ministry of Education.
316 MATERIALS AND METHODS
Sections
Specimens of kidney were obtained from rats with glomerulonephritis induced by bovine serum albumin (BSA) as previously described (Yamamoto et al., 1978). They were snap-frozen in liquid n-hexane at --70°C. Sections were made with a Minotome (Damon/IEC Division) by setting the scale at 1 and placed in a bath of cold 0.01 M phosphate-buffered saline, pH 7.2, (PBS) and air-dried. To evaluate the effect of conditions, specimens from rats with moderate or marked proliferative glomerulonephritis were used. Fresh rat s e r u m
Wistar rats were anesthetized with ether. Blood was collected by cutting the axillary vessels, allowed to clot at room temperature for 10 min and centrifuged at 4°C. When needed, serum was diluted with isotonic Veronalbuffered saline, pH 7.6 containing 0.15 mM CaC12 and 0.5 mM MgC12 (VB). Heat-inactivated serum
Fresh rat serum was inactivated at 56 ° C for various periods of time. Anti-C3 treatment of serum
Antiserum for the third c o m p o n e n t of complement (C3) was prepared by the modified m e t h o d of Mardiney and Miiller-Eberhard (1965). A crude globulin fraction of anti-C3 was obtained by precipitation of the sera in 33% saturated a m m o n i u m sulfate at 4°C and dialysed against VB. The globulin fraction was adjusted by dilution with VB to a concentration of 38 mg/ml. As a control, normal rabbit sera were treated with the same procedure as anti-C3 sera. One volume of several dilutions with VB of anti-C3 globulin fraction or normal globulin fraction was mixed with 4 vol of fresh rat serum diluted 1/10 with VB and placed at 4°C for 48 h and then the precipitate was spun down. PMN were suspended in the supernatant and incubated with sections. PMN
PMN were obtained from the peritoneal cavity of Wistar rats by the technique of Hirsch and Church (1960). Ten milliliters of 0.1% glycogen was injected intraperitoneally and the exudate was collected 3 h later and centrifuged at 800 rev/min for 10 min. The cell pellet containing over 95% PMN was resuspended in serum or diluted serum to various densities. Incubation
The equipment used is shown in Fig. 1. A glass slide with 6 sections was covered with another glass slide with two ribbons of electric insulating tape stretched across the slide. The distance between the two glass slides was
317
electrical insulating tape I
glass~slide
,± 0.2 mm I.
JT
glass slide with sections PMN suspended in serum Fig. 1. The e q u i p m e n t used for i n c u b a t i o n .
approximately 0.2 mm. PMN suspension was discharged into the space with a Pasteur pipet and the equipment was placed at 37°C in'a moist atmosphere for 40 min in the standard assay and for various periods of time (10--60 min) to examine the effect of incubation time. Then, the slide with sections was placed in a bath of cold PBS, rinsed gently with PBS, fixed with ethanol and stained with hematoxylin and eosin. Assessmen t PMN on 60 glomeruli of moderate size were counted and the results represented the average number of PMN on a glomerulus. Immunofluorescent technique As reported previously (Yamamoto et al., 1978), the sections were stained with fluorescein isothiocyanate-labeled anti-BSA, anti-rat IgG, and anti-rat C3. The intensity and the a m o u n t of fluorescence were arbitrarily graded as --, +, ++, and +++. RESULTS
Standard procedure When PMN were suspended in fresh rat serum to a concentration of 2 × 107/ml and incubated with sections of rat BSA nephritis, PMN were observed microscopically on glomeruli with i m m u n e deposits and n o t other regions o f the sections. As seen in Fig. 2a and b, PMN attached to glomeruli were countable with ease and the calculation of PMN on a certain glomerulus in serial sections did n o t give much difference. Precise location of PMN was u n k n o w n , but suspected to be closely associated with immune complexes as defined by the immunofluorescent technique. Application to a group o f rat BSA-nephritis As revealed in Table 1, about a hundred PMN were attached to a severely
O
•
•
,
....
:
