Clin. exp. Immunol. (1975) 22, 102-111.
THE ROLE OF POLYMORPHONUCLEAR LEUCOCYTES IN THE AUTOLOGOUS PHASE OF NEPHROTOXIC NEPHRITIS P. F. NAISH, N. M. THOMSON, I. J. SIMPSON AND D. K. PETERS Departments of Medicine and Immunology, Royal Postgraduate Medical School, Du Cane Road, London (Received 6 March 1975) SUMMARY
The role of polymorphonuclear leucocytes (PMN) in the autologous phase of nephrotoxic nephritis (NTN) in the rabbit has been investigated. Depletion of circulating PMN by nitrogen mustard protected renal function and immunofluorescent examination showed reduction in glomerular fibrin deposition. Depletion of circulating PMN using a highly specific goat anti-PMN serum (APS) provided similar protection of renal function, highly significant reduction in proteinuria and histological and immunofluorescent examination showed reduced glomerular PMN infiltration, extracapillary cell proliferation and virtual absence of fibrin deposition. Although protection by nitrogen mustard may have been partly due to immunosuppression, no reduction in antibody response was detected in the APS-treated rabbits. The results implicate the polymorph as the principal injurious agent in this model of NTN, responsible directly or indirectly for both proteinuria and glomerular fibrin deposition.
INTRODUCTION Polymorphonuclear leucocytes accumulate at the sites of the antigen-antibody reaction in various types of allergic tissue injury. Vascular injury in the Arthus reaction and in the necrotizing arteritis of serum sickness is largely prevented by depletion of circulating neutrophils (Humphrey, 1955; Cochrane, Weigle & Dixon, 1959; Kniker & Cochrane, 1965). The immediate or heterologous phase of nephrotoxic nephritis is characterized by the accumulation of polymorphs within the glomeruli and proteinuria is substantially reduced in rabbits and rats by prior depletion of circulating polymorphs (Cochrane, Unanue & Dixon, 1965; Hawkins & Cochrane, 1968; Henson, 1972). Less obvious glomerular polymorph accumulation also occurs in the autologous stage of nephrotoxic nephritis (NTN) (Ehrich, Forman & Seifter, 1952) but the role of polymorphs in the autologous phase has not so far been investigated. We have therefore undertaken a series of experiments to determine the importance of polymorphs in this nephritis in rabbits. Correspondence: Dr N. M. Thomson, Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS.
102
Polymorphs in nephrotoxic nephritis
103
MATERIALS AND METHODS The experiments were done in series. In the first experiment polymorph depletion was achieved by nitrogen mustard (mustine hydrochloride, Boots); the finding that substantial protection occurred led us to undertake a second series of experiments in which a specific anti-polymorph serum was used. Animals. Male New Zealand white rabbits, weighing between 2 and 2 5 kg were used. The animals were fed on a normal water and pellet diet. Preparation, characterization and administration of the nephrotoxic sera (NTS). Two separate anti-rabbit glomerular basement membrane (GBM) sera (nephrotoxic sera) were prepared in sheep, by the injection of particulate rabbit GBM in FCA at 2-weekly intervals until high titres of antibody to rabbit GBM were obtained. The nephrotoxic sera were decomplemented by heating at 560C for 30 min and absorbed with normal rabbit erythrocytes and plasma. The two antisera were of similar nephrotoxicity and intravenous injection of 0-5-1-5 ml of either serum led to a severe crescentic nephritis within 12-14 days. In other experiments (Thomson, Simpson & Peters, 1975) it was found that an intravenous dose of 1 ml of the second nephrotoxic antiserum resulted in a mean deposition of 180 pg of sheep antibody per kidney. Preparation ofgoat anti-rabbit polymorph serum (APS). An APS was prepared by the method of Kniker & Cochrane (1965). Briefly, 5 x 108 rabbit polymorphs in FCA obtained from the peritoneal cavity 4 hr after the instillation of 0-1y% rabbit glycogen, were injected into a goat on five occasions, at 2-weekly intervals. The antiserum was extensively absorbed against rabbit erythrocytes, platelets, lymphocytes and plasma. The gamma-globulin fraction was obtained by salt fractionation. Intravenous injection of 5 ml of this serum depleted circulating polymorphs for 18-24 hr. Blood and urine samples. Serum concentrations of urea and creatinine were determined by the autoanalyser method, C3 concentrations by radial immunodiffusion (Mancini, Carbonara & Heremans, 1965) and urine protein by the Biuret method. Peripheral blood white cell counts and platelet counts were estimated either by a manual counting chamber or Coulter counter. Differential white cell counts were assessed on a MayGrunwald-Giemsa stained smear. Circulating antibodies to sheep immunoglobulin were measured by a haemagglutination technique employing 1% ox red cells coated with sheep anti-ox red cell antibody at a subagglutinating titre, the system being stabilized with 200 normal rabbit serum. Histological and immunofluorescent preparation. Portions of kidney were fixed in 10%4 buffered formalin, sectioned at 4 pm and stained with H & E and PAS. Specimens were also snap-frozen in liquid nitrogen, sectioned at 4 um on a cryostat and stained with fluorescein isothiocyanate (FITC) conjugated antisera to sheep IgG, rabbit IgG, rabbit C3 and rabbit fibrin. All antisera were prepared in our laboratory and conjugated with FITC as described by Fothergill (1969). Assessment ofthe histological and immunofluorescent preparations. Sections were coded and analysis based on independent assessment. As described previously (Naish et al., 1972), extracapillary cell proliferation was quantitated by a scoring system in which each glomerulus was graded from 0 to 3 and the mean per cent incidence of each grade calculated. Glomerular fibrin deposition was scored similarly by immunofluorescence. At least fifty glomeruli per animal were examined by light microscopy and thirty by immunofluorescence using a Leitz Orthoplan system with a high pressure mercury light source (HBO-200), K-500 FITC interference filter, BG-38 red suppression and K-510 or K-530 barrier filters. Experimental protocol. Two series of experiments were performed. In the first polymorphs were depleted by nitrogen mustard (mustine series) and in the second anti-polymorph serum was used (APS series). Mustine-series. Thirty-one rabbits were injected intravenously on day 0 with NTS A. Seventeen of these animals received nitrogen mustard at a dose of 2 mg/kg, five on day 2 and twelve on day 4. The remaining fourteen control animals received saline. Animals were killed on day 10. APS-series. Twenty-one rabbits were injected intravenously with 1 ml/kg of NTS B. Five of these received anti-polymorph serum, 5 ml being injected intravenously daily, from day 4. On days 10 and 11 two doses of 5 ml were given. Five control animals received the same volume of normal goat serum daily. Eleven other control animals received 5 ml of saline intravenously daily. All animals in this series received prophylactic intravenous antibiotics (penicillin 20,000 u and streptomycin 50 mg) daily from day 4. Animals were killed on day 12. In both series of experiments serum urea, creatinine and C3 concentrations were estimated on alternate days. Proteinuria was quantitated on urine samples obtained daily by urethral catheterization in the APS series. Peripheral white cell counts, differential cell counts and platelet counts were estimated daily in both groups, extra estimations being carried out in the APS series at various times after the dose of APS.
P. F. Naish et al.
104
Statistical analysis. Comparison of the results obtained in the groups of animals was made using the Wilcoxon rank test for two samples (Wilcoxon, 1945).
RESULTS No deaths occurred in either series. The administration of the first and second doses of APS were associated with some peripheral vasocontriction lasting 6-12 hr but did not affect urine output. This was presumed to be the consequence of intravascular release of polymorph constituents before persistent polymorph depletion was achieved. On days 10 and 11, animals receiving APS and the control group receiving normal goat serum developed minor anaphylactic reactions.
Polymorph depletion Mustine series (Fig. la). The lowest total white cell count occurred 72 hr after the nitrogen mustard injection. There was not a large fluctuation in white cell counts in untreated animals at any stage of the experiment. 12
(a)
10
2
(b)
E8
_E
0 C 0
=4-
2-
(-and a) g~~~rous 0
2
4
6
8
10 0
2
4
6 8
10 12
Days
FIG. 1. Peripheral blood white cell counts. (a) Mustine series: mean total white cell counts for control (.), NM day 2 (in), and NM day 4 (A) groups. (b) APS series: mean polymorph (solid symbols) and lymphocyte (open symbols) counts for control (6 and 0) and APS-treated (U and o) groups.
APS series (Fig. lb). Immediate and sustained polymorph depletion was obtained after the injection of antipolymorph serum. Mean polymorph counts were less than 400/mm3 at all times except in two animals in which counts of 450 and 500/mm3 were recorded on days 10 and 11. In contrast, control animals showed a mild polymorphonuclear leukocytosis. Lymphocyte counts were never reduced by more than 15% after APS administration.
Platelet counts No significant reduction in platelet counts occurred after nitrogen mustard. APS caused a slight reduction in platelet counts but this was never greater than 10%.
105
Polymorphs in nephrotoxic nephritis
Rabbit anti sheep immunoglobulin antibody titres Mustine-series. In animals treated on day 4, antibody titres were significantly lower (P< 001) than in untreated animals on all days tested. The titres were also significantly lower (PO 1). Renal function Mustine series. Serum urea concentrations rose progressively in untreated animals from day 4 onwards (Table a). Values were significantly lower on day 8 in both groups of TABLE 1. (a) Serum urea concentrations in mustine series
Serum urea concentration (mg/100 ml): mean (range) Animal group
n
Controls
14
Mustine treated: Day 2
Day 4
5 12
Day 4
Day 6
Day 8
Day 10
38
41
(26-97)
60 (31-106)
144
(28-62)
(47-314)
215 (43-528)
Day 0
51*
41
59
90
(33-51)
(38-102)
(38-206)
(20-79) 49* (29-135)
44
44
53
(35-56)
(29-69)
(36-78)
109 (23-210) 46* (26-78)
P. F. Naish et al.
106
TABLE 1. (b) Serum creatine concentrations in APS series
Serum creatinine concentration (mg/100 ml): mean (range) Animal group Controls: Goat serum Saline APS-treated
n
Day 0
Day4
Day 6
Day 8
Day 10
Day 12
5
0-88 (08-10) 10
1.1 (09-1 3) 1.1
(0-8-1-7)
0 90
09
5 94 (39-7-2) 4-5 (3 5-8 5) 1-82*
7-3 (5-8-80)
(0-8-1-3)
1-86 (0-9-4-5) 2-2 (0 9-4 4) 1-56
(1.1-201)
(1-42-7)
11 5
(0-7-1-2)
(0-7-1*0) * P
THE ROLE OF POLYMORPHONUCLEAR LEUCOCYTES IN THE AUTOLOGOUS PHASE OF NEPHROTOXIC NEPHRITIS P. F. NAISH, N. M. THOMSON, I. J. SIMPSON AND D. K. PETERS Departments of Medicine and Immunology, Royal Postgraduate Medical School, Du Cane Road, London (Received 6 March 1975) SUMMARY
The role of polymorphonuclear leucocytes (PMN) in the autologous phase of nephrotoxic nephritis (NTN) in the rabbit has been investigated. Depletion of circulating PMN by nitrogen mustard protected renal function and immunofluorescent examination showed reduction in glomerular fibrin deposition. Depletion of circulating PMN using a highly specific goat anti-PMN serum (APS) provided similar protection of renal function, highly significant reduction in proteinuria and histological and immunofluorescent examination showed reduced glomerular PMN infiltration, extracapillary cell proliferation and virtual absence of fibrin deposition. Although protection by nitrogen mustard may have been partly due to immunosuppression, no reduction in antibody response was detected in the APS-treated rabbits. The results implicate the polymorph as the principal injurious agent in this model of NTN, responsible directly or indirectly for both proteinuria and glomerular fibrin deposition.
INTRODUCTION Polymorphonuclear leucocytes accumulate at the sites of the antigen-antibody reaction in various types of allergic tissue injury. Vascular injury in the Arthus reaction and in the necrotizing arteritis of serum sickness is largely prevented by depletion of circulating neutrophils (Humphrey, 1955; Cochrane, Weigle & Dixon, 1959; Kniker & Cochrane, 1965). The immediate or heterologous phase of nephrotoxic nephritis is characterized by the accumulation of polymorphs within the glomeruli and proteinuria is substantially reduced in rabbits and rats by prior depletion of circulating polymorphs (Cochrane, Unanue & Dixon, 1965; Hawkins & Cochrane, 1968; Henson, 1972). Less obvious glomerular polymorph accumulation also occurs in the autologous stage of nephrotoxic nephritis (NTN) (Ehrich, Forman & Seifter, 1952) but the role of polymorphs in the autologous phase has not so far been investigated. We have therefore undertaken a series of experiments to determine the importance of polymorphs in this nephritis in rabbits. Correspondence: Dr N. M. Thomson, Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS.
102
Polymorphs in nephrotoxic nephritis
103
MATERIALS AND METHODS The experiments were done in series. In the first experiment polymorph depletion was achieved by nitrogen mustard (mustine hydrochloride, Boots); the finding that substantial protection occurred led us to undertake a second series of experiments in which a specific anti-polymorph serum was used. Animals. Male New Zealand white rabbits, weighing between 2 and 2 5 kg were used. The animals were fed on a normal water and pellet diet. Preparation, characterization and administration of the nephrotoxic sera (NTS). Two separate anti-rabbit glomerular basement membrane (GBM) sera (nephrotoxic sera) were prepared in sheep, by the injection of particulate rabbit GBM in FCA at 2-weekly intervals until high titres of antibody to rabbit GBM were obtained. The nephrotoxic sera were decomplemented by heating at 560C for 30 min and absorbed with normal rabbit erythrocytes and plasma. The two antisera were of similar nephrotoxicity and intravenous injection of 0-5-1-5 ml of either serum led to a severe crescentic nephritis within 12-14 days. In other experiments (Thomson, Simpson & Peters, 1975) it was found that an intravenous dose of 1 ml of the second nephrotoxic antiserum resulted in a mean deposition of 180 pg of sheep antibody per kidney. Preparation ofgoat anti-rabbit polymorph serum (APS). An APS was prepared by the method of Kniker & Cochrane (1965). Briefly, 5 x 108 rabbit polymorphs in FCA obtained from the peritoneal cavity 4 hr after the instillation of 0-1y% rabbit glycogen, were injected into a goat on five occasions, at 2-weekly intervals. The antiserum was extensively absorbed against rabbit erythrocytes, platelets, lymphocytes and plasma. The gamma-globulin fraction was obtained by salt fractionation. Intravenous injection of 5 ml of this serum depleted circulating polymorphs for 18-24 hr. Blood and urine samples. Serum concentrations of urea and creatinine were determined by the autoanalyser method, C3 concentrations by radial immunodiffusion (Mancini, Carbonara & Heremans, 1965) and urine protein by the Biuret method. Peripheral blood white cell counts and platelet counts were estimated either by a manual counting chamber or Coulter counter. Differential white cell counts were assessed on a MayGrunwald-Giemsa stained smear. Circulating antibodies to sheep immunoglobulin were measured by a haemagglutination technique employing 1% ox red cells coated with sheep anti-ox red cell antibody at a subagglutinating titre, the system being stabilized with 200 normal rabbit serum. Histological and immunofluorescent preparation. Portions of kidney were fixed in 10%4 buffered formalin, sectioned at 4 pm and stained with H & E and PAS. Specimens were also snap-frozen in liquid nitrogen, sectioned at 4 um on a cryostat and stained with fluorescein isothiocyanate (FITC) conjugated antisera to sheep IgG, rabbit IgG, rabbit C3 and rabbit fibrin. All antisera were prepared in our laboratory and conjugated with FITC as described by Fothergill (1969). Assessment ofthe histological and immunofluorescent preparations. Sections were coded and analysis based on independent assessment. As described previously (Naish et al., 1972), extracapillary cell proliferation was quantitated by a scoring system in which each glomerulus was graded from 0 to 3 and the mean per cent incidence of each grade calculated. Glomerular fibrin deposition was scored similarly by immunofluorescence. At least fifty glomeruli per animal were examined by light microscopy and thirty by immunofluorescence using a Leitz Orthoplan system with a high pressure mercury light source (HBO-200), K-500 FITC interference filter, BG-38 red suppression and K-510 or K-530 barrier filters. Experimental protocol. Two series of experiments were performed. In the first polymorphs were depleted by nitrogen mustard (mustine series) and in the second anti-polymorph serum was used (APS series). Mustine-series. Thirty-one rabbits were injected intravenously on day 0 with NTS A. Seventeen of these animals received nitrogen mustard at a dose of 2 mg/kg, five on day 2 and twelve on day 4. The remaining fourteen control animals received saline. Animals were killed on day 10. APS-series. Twenty-one rabbits were injected intravenously with 1 ml/kg of NTS B. Five of these received anti-polymorph serum, 5 ml being injected intravenously daily, from day 4. On days 10 and 11 two doses of 5 ml were given. Five control animals received the same volume of normal goat serum daily. Eleven other control animals received 5 ml of saline intravenously daily. All animals in this series received prophylactic intravenous antibiotics (penicillin 20,000 u and streptomycin 50 mg) daily from day 4. Animals were killed on day 12. In both series of experiments serum urea, creatinine and C3 concentrations were estimated on alternate days. Proteinuria was quantitated on urine samples obtained daily by urethral catheterization in the APS series. Peripheral white cell counts, differential cell counts and platelet counts were estimated daily in both groups, extra estimations being carried out in the APS series at various times after the dose of APS.
P. F. Naish et al.
104
Statistical analysis. Comparison of the results obtained in the groups of animals was made using the Wilcoxon rank test for two samples (Wilcoxon, 1945).
RESULTS No deaths occurred in either series. The administration of the first and second doses of APS were associated with some peripheral vasocontriction lasting 6-12 hr but did not affect urine output. This was presumed to be the consequence of intravascular release of polymorph constituents before persistent polymorph depletion was achieved. On days 10 and 11, animals receiving APS and the control group receiving normal goat serum developed minor anaphylactic reactions.
Polymorph depletion Mustine series (Fig. la). The lowest total white cell count occurred 72 hr after the nitrogen mustard injection. There was not a large fluctuation in white cell counts in untreated animals at any stage of the experiment. 12
(a)
10
2
(b)
E8
_E
0 C 0
=4-
2-
(-and a) g~~~rous 0
2
4
6
8
10 0
2
4
6 8
10 12
Days
FIG. 1. Peripheral blood white cell counts. (a) Mustine series: mean total white cell counts for control (.), NM day 2 (in), and NM day 4 (A) groups. (b) APS series: mean polymorph (solid symbols) and lymphocyte (open symbols) counts for control (6 and 0) and APS-treated (U and o) groups.
APS series (Fig. lb). Immediate and sustained polymorph depletion was obtained after the injection of antipolymorph serum. Mean polymorph counts were less than 400/mm3 at all times except in two animals in which counts of 450 and 500/mm3 were recorded on days 10 and 11. In contrast, control animals showed a mild polymorphonuclear leukocytosis. Lymphocyte counts were never reduced by more than 15% after APS administration.
Platelet counts No significant reduction in platelet counts occurred after nitrogen mustard. APS caused a slight reduction in platelet counts but this was never greater than 10%.
105
Polymorphs in nephrotoxic nephritis
Rabbit anti sheep immunoglobulin antibody titres Mustine-series. In animals treated on day 4, antibody titres were significantly lower (P< 001) than in untreated animals on all days tested. The titres were also significantly lower (PO 1). Renal function Mustine series. Serum urea concentrations rose progressively in untreated animals from day 4 onwards (Table a). Values were significantly lower on day 8 in both groups of TABLE 1. (a) Serum urea concentrations in mustine series
Serum urea concentration (mg/100 ml): mean (range) Animal group
n
Controls
14
Mustine treated: Day 2
Day 4
5 12
Day 4
Day 6
Day 8
Day 10
38
41
(26-97)
60 (31-106)
144
(28-62)
(47-314)
215 (43-528)
Day 0
51*
41
59
90
(33-51)
(38-102)
(38-206)
(20-79) 49* (29-135)
44
44
53
(35-56)
(29-69)
(36-78)
109 (23-210) 46* (26-78)
P. F. Naish et al.
106
TABLE 1. (b) Serum creatine concentrations in APS series
Serum creatinine concentration (mg/100 ml): mean (range) Animal group Controls: Goat serum Saline APS-treated
n
Day 0
Day4
Day 6
Day 8
Day 10
Day 12
5
0-88 (08-10) 10
1.1 (09-1 3) 1.1
(0-8-1-7)
0 90
09
5 94 (39-7-2) 4-5 (3 5-8 5) 1-82*
7-3 (5-8-80)
(0-8-1-3)
1-86 (0-9-4-5) 2-2 (0 9-4 4) 1-56
(1.1-201)
(1-42-7)
11 5
(0-7-1-2)
(0-7-1*0) * P
