Circulating immune complexes and the nephropathy of cystic fibrosis

Circulating Immune Complexes and the Nephropathy of Cystic Fibrosis CHARLESA. DAVIS, MD, CARLOS R. ABRAMOWSKY, MD, AND GARY SWINEHART, BA To explore the putative nephropathic role of Pseudomonas-associated immune complexes, the authors measured the quantity of immune complexes in sera obtained, before death, from 20 patients with cystic fibrosis, and compared these findings with the histologic features of the lesions and with immunofluorescence patterns of kidney tissue obtained at autopsy.The immune complexes were measured by solid-phase Clq (Clq immunecomplex) and conglutinin to detect complexes containing IgM, IgA, and IgG. Elevated levels of Clq immune complex (13 patients) suggested the possibility of renal deposition of C3 (P < 0.005) and IgM (P < 0.05). The only three patients with IgA tissue deposits had elevated levels of Clq immune complex with normal IgA immune complexes. No other assay findings correlated with the immunofluorescencefindings. Despite the prominent C3 in tissue deposits, the histologic features were not sig* nificantly associated with the results of the immune complex assays. This study indicates that complement-activatingIgMcontaining complexes can be deposited in renal tissues of patients with cystic fibrosis, but their nephropathogenicity is doubtful. These observations of kidney lesions, which diminish the injurious role of immune complexes in cystic fibrosis, may be relevant to an understanding of the pathogenesis of the lung lesions, which recent studies have linked to the presence of immune complexes. HuM PATttOL 15:244--247, 1984.

Circulating immtme complexes in cystic fibrosis have been demonstrated by a variety of methods, 1-4 but their significance is unclear, particularly in view of tile paucity of extrapulmonary signs of immune complex disease. Indirect evidence of a pathogenic role for these complexes has been provided recently4,5 by the demonstration of their rising levels in association with exacerbations of pulmonary symptoms and by the demonstration of immunoglobulin deposits in the spleen, liver, thymus, pancreas, and intestines of patients with cystic fibrosis. 6 In a study of several potentially nephrotoxic factors in cystic fibrosis, Abramowsky and Swinehart 7 provided immunofluorescent and ultrastructural evidence suggesting that immune complexes probably containing pseudomonas antigen might injure the kidney. To evaluate the role of these complexes in causing renal injury in patients with cystic fibrosis and to gain insight into their possible role in lung damage, we compared renal histologic findings and immunofluorescent deposits in autopsy tissue with immune

Received from the Departments of Pediatrics and Pathology, University Hospitalsof Clevelandand Case Western Reserve University School of Medicine, Cleveland. Ohio. Accepted for publication March 18, 1983. Address correspondence and reprint requests to Dr. Abramowsky: Institute of Pathology,Case Western ReserveUniversity, 2085 Adelbert Rd, Cleveland,OH 44106.

244

complex levels in antemortem sera from patients with cystic fibrosis.

MATERIALSAND METHODS The group consisted of 20 patients (nine male, 11 female) with cystic fibrosis, whose ages ranged from 12 to 34 years (mean, 21 )'ears). Renal tissues were obtained within three hours of death, and finorescence studies were performed on frozen sections stained with antibodies to IgG, IgA, IgM, C3, properdin, and C lq as previously described. 7 Histologic sections stained with h e m a t o x y l i n - e o s i n , periodic a c i d - S c h i f f (PAS), Jones' stain, and Masson's trichrome were evaluated by the method of Pirani and Salinas-Madrigal, s in which individual lesions of the renal parenchyma are graded semi-quantitatively on a scale from 0 to 4 + . On the basis of our previously described lesions, 7 we evaluated glomerular proliferation and sclerosis, tubulointerstitial disease, and vascular lesions. To relate the parenchymal damage more realistically to tile presence of immune complexes, acute tubular necrosis, hyperplasia of thejuxtaglomerular apparatus, and glomerulomegaly, also previously described, 7 were exchtded from evaluation. The sum of all gradings front each kidney was designated the histopathology score, and was considered an expression o f intensity o f renal tissue damage. For immune assays, sera were obtained before death in conjunction with other studies and in compliance with our review board rules. All sera were stored frozen at - 7 0 ~ until used. The Clq immune complex assay9,10 and the conglutinin assays ~1 were modified to a microtiter technique. 12 In the C l q immune complex assay, microtiter wells (Cook Engineering Co., Alexandria, Virginia) were coated with 0.1 ml of Clq (1 ~g/ml)13; 0.005-ml serum samples were added to 0.095 ml of buffer, and the previously described 9 37~ incubations were lengthened from 60 to 90 minutes. Modifications of the conglutinin assay included coating of microtiter wells with 0.1 ml of bovine conglutinin (10 Isg/ml), 14 adding 0.1 per cent Tween-20 to all buffers after the wells had been coated, using 0.01 ml of serum for every 0.09 ml of buffer, and incubating the samples in conglutinin-coated wells for 90 minutes at 37~ and then overnight at 4~ Protein A (Pharmacia, Piscataway, New Jersey), monoclonal antihuman IgA and IgM (Bethesda Research Laboratories, Gaithersburg, Maryland), and monoclonal a n t i h u m a n IgG (supplied by Michael

CIRCULATINGIMMUNECOMPLEXESIN CYSTICFIBROSIS(Davis et al.)

Fanger, MD, Case Western Reserve University) were radiolabeled by the lactoperoxidase method. 15 Standards for the protein A and anti-IgG assays consisted of human IgG (Miles Laboratories, Elkhart, Indiana), which was heat aggregated at 60~ for 30 minutes, diluted in normal human serum, and incubated at 37~ for 30 minutes. Results o f the anti-IgG and Clq assays were expressed in micrograms per milliliter, and results of the anti-IgA and anti-IgM assays were expressed as percentages o f the mean counts per minute for sera obtained f r o m ten normal adults. Values more than two standard deviations above the mean were considered abnormal. These assays, as modified, have been tested in our laboratory in normal subjects and in patients with systemic lupus erythematosus a n d other immunecomplex-related diseases and found to be as reliable as the standard techniques (C. A. Davis, unpublished data).

40.

35"

30"

A

250

25,

e

~2oo-

::" 20-

U

15-

o

II iii III

Iso. . . . . -*:---

Qo

u I0'

eo oll

I

o mlB

245

o

O Z

RESULTS

In 11 of 13 patients with elevated levels of serum immune complexes found on the Clq assay (fig. 1; table 1), C3 deposits were revealed by fluorescence. O f these, ten had associated immunoglobulin deposits, including IgM. T h e seven patients with n o r m a l s e r u m levels o f C l q i m m u n e complex included two with immunoglobulin deposits only and five with negative fluorescence. By chi-square analysis, these results showed a significant positive correlation between elevated levels of serum C l q immune complex and tissue deposits of C3 (P < 0.005) and a less significant correlation with tissue deposits of IgM (P < 0.05). Ten of the 20 patients had elevated levels of serum IgM complexes; of these, eight showed IgM tissue deposits (seven with C3). Of the other ten patients with normal IgM serum complexes, four had IgM in their glomeruli. These results indicate a tendency of IgM deposition in tissues to reflect an elevation of serum IgM-containing complexes. However, this tendency was not significant and may have been influenced by the associated C3. Tissue deposits of IgA were found in three patients, all of whom had correspondingly elevated C 1q i m m u n e complex but normal serum IgA complex levels (fig. 1). Specimens assayed for IgG immune complexes showed no correlation with their corresponding tissue immunofluorescence patterns. As in our previous study, 7 a few specimens showed Clq and properdin tissue deposits (table 1). T h e histopathologic changes in the kidneys were similar to those previously reported. 7 The histopathology scores were compared only with the corresponding serum levels of Clq and IgM complexes, since they were the only ones that correlated with C3 and IgM tissue deposits. No association was found between Clq or IgM serum immune complex levels and histologic lesions, regardless o f w h e t h e r the overall histopathology score or the lesions of individual renal parenchymal structures were evaluated.

I

I,.

U

.....

u

,.o loo-

.o-o-...

:

,..:

,,g

....

oo oO~r oiL._.

"tlg 0 e

0

50e

0 IImom

0

IgG

Ciq

IgA

IgM

FIGURE t. Circulating immune complex [ClC) levels as determined by various assays and immunofluorescence results. Levels of

Clq and IgG complexes are shown on the left graph; levels of IgA and IgM complexes are shown on the right. The IgA and IgM levels are expressed as percentages of the means established in ten normal subjects [see text]. The dotted line on each graph represents the upper level of normal [ + 2 SD).The renal immunofluorescence findings for each sample are represented as follows: solid circle, negative immunofluorescence; open circle, immunoglobulins and C3; solid triangle, immunoglobulins only, solid square, C3 only.

DISCUSSION

I m m u n e complex levels have been found to be elevated in the sera of patients with cystic fibrosis, as d e t e r m i n e d by several assays. 1-5 In these studies, more than 50 per cent of the patients had elevated serum i m m u n e complexes; the higher levels were found in patients with clinically exacerbated hmg disease. These observations, coupled with the demonstration of antibodies to Pseudomonas aeruginosa in the isolated complexes 16 and the presence of fluorescent deposits in lungs 6 have pointed to immune complexes as being at least partially responsible for lung damage in cystic fibrosis. In a previous study of tile kidney in cystic fibrosis, we explored several factors potentially responsible for the chronic renal damage. 7 In addition to aminoglycosides, hypoxia, and heart failure, among others, we suggested i m m u n e complex damage to tile kidney as a possible mechanism of nephrotoxicity in cystic fibrosis. This suggestion was based on the observation of IgM and C3 deposits and electron-dense deposits in the glomeruli. To further evaluate the potential role of immune complexes in producing tissue injury, we compared the pattern of immtme deposits in kidneys obtained at autopsy from 20 patients with cystic fibrosis with

HUMANPATHOLOGY Volume15, No. 3 (March 1984l

TABLE 1. Renal Fluorescence, Serum Immune Complex Levels, and Histopathology Scores for 20 Patients with Cystic Fibrosis Age (}ears), Sex Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient

1" 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

18, 26, 24, 19, 17, 19, 12, 15, 16, 26, 18, 34, 28, 20, 19, 15, 26, 25, 22, 27,

M M F M M F F F F M M M F F F F M M F F

Renal h n m u n o f l u o r e s c e n c e IgG

lgM

IgA

C3

C]q

+ + + . . . + . . . .

+ + + + + +

+ +

+ + + + + + . + . + + + + .

+ + . + . + . . . . . . .

.

. +

.

. + + +

.

. + -

. + +

. . . .

. -

. . .

. . . .

. . . . . .

. . . . . .

Properdin + + + + -

Immune Complexest Czq (p.g/ml) 38 32.0 29 26 24 15.5 13.5 13.5 12.0 9.0 9.0 7.6 7.6 6.8 6.5 6.2 3.8 2.3
IgM (% normal mean)

lgA (ok normal mean}

IgG (~g/ml)

Histopathology Score

145 127 182 111 145 98 168 138 72 160 80 134 64 116 135 119 126 116 113 94

99 156 113 169 78 84 197 169 139 120 169 119 83 193 75 230 105 151 119 83

27 ND 9.9 3.6 <1.5 <1.5 5.8 <1.5 3.0 ND <1.5 <1.5 ND <1.5 <1.5 7.1 ND 6.3 9.5 ND

13 15 11 7 16 17 13 5 3 8 11 21 9 9 14 13 8 26 8 9

ABBREVIATION: ND, not done. * Patients 1 t h r o u g h 13 had elevated levels o f s e r u m C l q i m m u n e complexes. ? U p p e r limits o f n o r m a l (mean + 2 SD): C l q , 7.5 ~g/ml; IgM, 135% o f n o r m a l mean; lgA, 150% o f normal m e a n ; IgG, 50 ~tg/ml

the corresponding levels of immune complexes measured in antemortem sera. We also graded renal histologic lesions semi-quantitatively in order to assess the degree of pathogenicity of the complexes. These studies showed a high positive correlation between tissue deposits of C3 and IgM and elevated levels of Clq immune complex in the serum. The preponderance of IgM in tissue deposits finds a parallel in the report of Schiotz et al., l who found IgM and, less f r e q u e n t l y C3, by fluorescence techniques in the d e r m o - e p i d e r m a l junction in patients with cystic fibrosis. Curiously, in the same study 13 of 21 patients showed serum antinuclear antibodies predominating in the IgM class. Immunoglobulin A was found in the kidneys of three of our patients, all of whom had normal levels o f IgA complexes but abnormal levels o f Clq inamune complex. A possibly related finding is the observation by Moss and Lewiston 3 of elevated serum IgA in patients with high Clq complexes. In this study we found no evidence of enhanced histologic damage in any compartment o f renal parenchyma in the group o f patients with high Clq imm u n e complex. However, in our previous study, 7 renal histologic vascular lesions were seen most often in specimens with positive immunofluorescence. Despite the prominence of C3 in our renal tissue studies and the elevated Clq immune complex, the role o f complement in causing tissue injury in patients with cystic fibrosis remains controversial. In the recent study by C h u r c h et al., 4 se~'um C3 and C4 levels had no relation to immune complex levels or to the clinical condition of the patients. This apparent lack of correlation between renal injury and comple246

m e n t deposition defies one o f tile basic tenets ot modern immunopathology. However, current studies t7 have indicated that, although complement remains a pivotal effector of tissue damage, other mediators, derived from neutrophils, monocytes, and soluble factors, are crucial. It remains to be seen whether these additional mechanisms are operational in patients with cystic fibrosis. On the basis of these observations, we feel that immune complexes play a minor role in the pathogenesis of the renal lesions of cystic fibrosis previously described by us. Since immtme complexes have been implicated to some extent in the pathogenesis of hmg damage in cystic fibrosis,4, 5 it is i m p o r t a n t to d e t e r m i n e , through future studies, whether our findings in the kidney have any bearing on the possible pathogenicity of these complexes in the lung.

REFERENCES 1. Schiotz PO, Hoiby N , J u l d F, et al: I m m u n e complexes in cystic fibrosis. Acta Pathol Microbiol Scand [C] 85:57, 1977 2. Berdischewsky M, Pollack M, Y o u n g LS, et al: Circulating imm u n e complexes in cystic fibrosis. Pediatr Res 14:830, 1980 3. Moss RB, Lewiston NJ: I m m u n e complexes a n d I m m o r a l response to Pseudomonas aeruginosa in cystic fibrosis. A m Rev Respir Dis 121:23, 1980 4. C h u r c h JA, J o r d a n SC, Keens TG, et ah Circulating i m m u n e complexes in patients with cystic fibrosis. Chest 80:405, 1981 5. Moss RB, H s u YP, Lewiston NJ: 123I-Clq-binding and specific antibodies as indicators o f p u l m o n a r y disease activity in cystic fibrosis. J Pediatr 99:215, 1981 6. McFarlane H, Holzel A, Brenchley P, et al: I m m u n e complexes in cystic fibrosis. Br Med J 1:423, 1975

CIRCULATINGIMMUNECOMPLEXESIN CYSTICFIBROSIS[Davis et al.]

12. Singh VK, Tingle AJ: Detection of circulating immune complexes by a Clq-microplate ELISA system. J Immunol Methods 50:109, 1982 13. Tenner AJ, Lesavre PH, Cooper NR: Purification and radiolabeling of human Clq. J Immunol 127:640, 1981 14. Lachman PM, Hobart JJ: Complement technology. In Weir DM (ed): ttandbook of Experimental Immunology, vol 1. Oxford, Blackwetl Scientific Pub, 1978, p 5A.I 15. David GS: Solid state lactoperoxidase: a highly stable enzyme for simple, gentle iodination of proteins. Biochem Biophys Res Commun 48:464, 1972 16. Moss RB, Hsu YP: Isolation and characterization of circulating immune complexes in cystic fibrosis. Clin Exp Immunol 47:301, 1982 17. Johnson KJ, Ward PA: Newer concepts in the pathogenesis of immune complex-induced tissue injury. Lab Invest 47:218, 1982

7. Abramowsky CR, Swinehart GL: The nephropathy of cystic fibrosis: a human model of chronic nephrotoxicity. HUM PATHOL 13:934, 1982 8. Pirani CL, Salinas-Madrigal L: Evaluation of percutaneous renal biopsy. Pathol Annu 3:253, 1968 9. Davis CA, Marder H, West CD: Circulating immune complexes in membranoproliferative glonaerulonephritis. Kidney Int 20:728, 1981 10. Ha}' FC, Nineham LJ, Roitt hM: Routine assay for the detection of immune complexes of known immunoglobulinclass using solid phase Clq. Clin Exp hnmunol 24:396, 1976 11. Casali P, Bossus A, Carpenter NA, et al: Solid-phase enzyme immunoassay and radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-bindingtest. A cmnparative study with 1251-labelled Clq binding and Raji-cell tests. Clin Exp Immunol 29:342, 1977

247