Clin. exp. Immunol (1975) 21, 75-81.

IgG AND IgA ANTIGENS IN HUMAN RENAL BASEMENT MEMBRANES J. N. McCORMICK AND W. PAGE FAULK Rheumatic Diseases Unit, Northern General Hospital, Edinburgh, and MRC Rheumatism Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire (Received 21 November 1974) SUMMARY

Cryostat sections of fresh human kidney studied by immunofluorescence were negative for IgG, IgA, IgM, Fc and Fab fragments of IgG, and kappa and lambda light chains. All of these conjugates except anti-IgM stained glomerular basement membranes and several other connective tissues if the cryostat sections were washed in phosphate-buffered saline (PBS) before being studied. Staining was inhibited by blocking reactions as well as by fixation before washing in PBS or by washing in non-ionized or hypotonic solutions. Results of several other aspects of this reaction are also reported. INTRODUCTION Immunofluorescence examination of renal biopsy specimens is widely employed as a diagnostic aid in investigations of human kidney diseases. The presence of staining and the pattern of immunofluorescence observed often help to establish a diagnosis. In order to differentiate between passive adsorption of serum IgG in tissue and IgG specifically deposited by immune mechanisms, biopsy sections are commonly washed before staining. The effects of such washing procedures are not known, but it has been suggested that they affect subsequent staining reactions (Holborow, 1971; Faulk & Hijmans, 1972; Weir, 1973). In this paper the results of staining washed and unwashed kidney sections are compared, and we report the artefactual exposure of IgG and IgA antigens in human renal basement membranes by washing in phosphate-buffered saline (PBS).

MATERIALS AND METHODS Tissues

Specimens of human kidney were obtained at autopsy from nine adults known not to have kidney disease and from two adults with rheumatoid arthritis. Renal biopsy specimens were Correspondence: Dr J. N. McCormick, Rheumatic Diseases Unit, Northern General Hospital, Edinburgh EH5 2DQ.

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obtained from fourteen children of a previously described group (Faulk et al., 1974) undergoing investigation in a paediatric renal unit. All tissues were snap-frozen in either isopentane cooled in liquid nitrogen or in a mixture of dry-ice and acetone. Antisera Immunochemically pure human y, a and u heavy chains and kappa and lambda light chains were prepared by previously described techniques (Wang et al., 1969; 1970), and these antigens were used to raise monospecific antisera in rabbits. The specificity of these antisera was monitored by haemagglutination and haemagglutination-inhibition experiments (Faulk & Houba, 1973). Immunoglobulin (Ig) enriched ammonium sulphate-prepared globulin fractions of each antiserum were labelled with isomer I fluorescein-isothiocyanate (FITC) (Baltimore Biological Laboratories, Baltimore, Maryland) by the dialysis technique and FITC-labelled IgG fractions were prepared from each conjugate by DEAE-cellulose chromatography as described by Faulk & Hijmans (1972). The fluorescein: protein ratios of these conjugates ranged from 1[7 to 2-8. In addition, FITC-labelled antisera specific for Fab and Fc fragments of human IgG and for monkey, rabbit, guinea-pig, swine, horse, cow, dog, goat, mouse, chicken, hamster, and rat Ig were obtained commercially (Nordic Immunological Laboratories, Tilburg, Netherlands).

Immunofluorescence techniques Pairs of sections cut at 4 pm in a cryostat were positioned 2-5 cm apart on a microscope slide and dried with an electric fan for 1 hr at room temperature. The slides were then placed for 30 min in a Coplin jar a third full of 0 01 M PBS, pH 7 2, so that the lower section was immersed and the upper section left dry. The slides were then drained, buffer was wiped off carefully around the lower section, both sections were stained in a moist chamber for 30 min with the appropriate FITC-labelled antiserum, washed three times in PBS for 20 min per wash, and mounted in 9000 glycerol-PBS. Some sections were fixed before washing in PBS and others were washed in buffers of varying pH, salt concentration, and temperature as detailed in Table 2 to determine the effect of these physical conditions on washed and unwashed sections. Preparations were examined with a Leitz Ortholux microscope equipped with an HBO 200 mercury lamp, UG5 15 mm exciter filter and K 460 barrier filter. Criteria of immunofluorescence specificity were established by inhibition and absorption techniques as described by McCormick et al. (1971).

RESULTS IgG antigens (a) Unwashed sections. In six of the fourteen renal biopsies, the anti-y chain conjugate produced bright linear or granular staining of the glomerular basement membrane (GBM) as anticipated from the clinical diagnosis. The remaining eight renal biopsies and all of the eleven postmortem kidneys were negative apart from a trace of specific fluorescence in the adventitia and lumen of some blood vessels (Fig. 1). (b) Washed sections. After washing in PBS, sections of all the renal biopsies and postmortem kidney specimens showed intense linear fluorescence on the glomerular and tubular basement membranes (BM) as well as distinct but less intense staining of peritubular reticu-

IgG and IgA in renal basement membranes

FIG. 1

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FIG. 2

FIG. 1. FITC-anti-IgG immunofluorescence of unwashed human kidney from patient without renal disease. Note that no fluorescence is seen. (The vascular adventitia and elastic laminae are autofluorescent.) This is a typical result of staining unwashed, undiseased, cryostat sections with FITC-anti-IgG. (Magnification x 150.) FIG. 2. FITC-anti-IgG immunofluorescence of washed human kidney from patient without renal disease. Note intense fluorescence of areas recorded in Table 1. This is a typical result of staining washed, undiseased, cryostat sections with FITC-anti-IgG. (Magnification x 150.)

lin, vascular adventitia and Bowman's capsule when exposed to the anti-y chain conjugate (Figs 2 and 3). An identical distribution of fluorescence was produced by the conjugates of rabbit antisera to human Fab, Fc, kappa and lambda chains, and the anti-monkey Ig conjugate. The labelled anti-rabbit Ig reacted with Bowman's capsule, tubule BM, and peritubular capillaries, and weakly with GBM and the adventitia of vessels, but the other antispecies Ig conjugates did not react with any of the connective tissue components (Table 1). Staining by the anti-y chain conjugate was inhibited by unlabelled anti-y chain serum but not by anti-c or anti-c chain sera. Staining was also abolished by absorbing the conjugate with purified IgG coated on chromic chloride-treated red cells (Faulk & Houba, 1973).

IgA antigens (a) Unwashed sections. In one of the renal biopsies specific fluorescence was seen on GBM

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FIG. 4 FIG. 3 FIG. 3. FITC-anti-IgG immunofluorescence of washed human kidney from patient without renal disease. Note linear fluorescence of GBM. This is a higher magnification of Fig. 2. (Magnification x 600.) FIG. 4. FITC-anti-IgA immunofluorescence of washed human kidney from patient without renal disease. Note 'dot-dash' staining of GBM. This is a typical result of staining washed, undiseased, cryostat sections with FITC-anti-IgA. Other results of anti-IgA staining are listed in Table 1. (Magnification x 1500.)

but all the other renal biopsies and post-mortem material were negative when exposed to the anti-a chain conjugate without prior washing in PBS. (b) Washed sections. All the biopsy and post-mortem specimens showed convincing staining of vascular adventitia, peritubular reticulin and GBM when exposed to the anti-cx chain conjugate after washing in PBS. The Bowman's capsule itself was unstained but was outlined by brightly stained reticulin fibres. Under high magnification the GBM staining presented a regular 'dot-dash' appearance (Fig. 4) which was quite different from the smooth, linear fluorescence observed with the anti-y chain conjugate (Fig. 3). The anti-a chain staining was inhibited by unlabelled anti-a chain serum but not by anti-y chain or anti-p chain serum and was abolished by absorption with purified IgA paraprotein.

IgG and IgA in renal basement membranes

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TABLE 1. Type and location of Ig antigens in human kidney detected by immunofluorescence following PBS wash

Location of antigens Types of antigens

Human IgG Human y-chain Human Fc (y) Human Fab (y) Human kappa Human lambda Human a-chain Human p-chain Monkey Ig Rabbit Ig

Glomerular BM

Bowman's capsule

Tubule

+ + + + + + +

+ + + + + +

+ (+)

+ +

Peritubular capillaries

Adventitia of vessels

+ + (+) + + +

+ + (+) + + + +

+ +

+ +

+ +

BM

Media of vessels

(+) + + + +

-

+ (+)

All staining reactions are negative in unwashed tissues. (+) Indicates a weakly positive reaction. FITC-labelled antisera to Ig from the following species were negative: guinea-pig; horse; rat; mouse; chicken; hamster; swine; cow.

IgM immunofluorescence IgM was found in the glomeruli of only two biopsy specimens before washing and did not appear in the others after washing in PBS. The results of all of these inimunofluorescence studies are summarized in Table 1.

Effect of salt concentration, pH, temperature and fixation (a) Salt concentration. When kidney sections were washed in isotonic non-ionized solutions such as 9.25% sucrose or in hypotonic saline solutions at a molarity of 0-038 or less, IgG and IgA immunofluorescence was not obtained (Table 2). (b) pH. The exposure of IgG and IgA antigens was not abolished by washing sections for 2 hr in isotonic saline buffered at pH 10'0-2-5 before rinsing briefly in PBS and staining with the appropriate conjugate (Table 2). (c) Temperature. To determine the effect of temperature, sections were immersed for 30 min in PBS maintained at temperatures ranging in 50C increments from 350C-650C. Staining of basement membrane and reticulin was absent following incubation at 650C and barely discernible at 60'C whereas washing at 350C-550C was no less effective in exposing IgG and IgA antigens than washing at room temperature (Table 2). (d) Fixation. The PBS phenomenon could not be demonstrated if sections were fixed in 9500 ethanol, formaldehyde or glutaraldehyde before washing in PBS. On the other hand, fixation in 9500 ethanol after washing in PBS and rinsing in distilled water did not abolish the PBS effect. Exposure of IgG and IgA antigens was also demonstrated in sections fixed in a mixture of 40 paraformaldehyde, 7.500 sucrose and 0 1 M phosphate buffer, pH 7-2, without prior washing in PBS.

J. N. McCormick and W. Page Faulk

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TABLE 2. Effect of pH, salt concentration and temperature on immunofluorescence staining for IgG in human kidney following PBS wash pH

NaCi concentration

Sucrose

Temperature (0C)

IgG staining

(M)

+ + + + + + + + (+)

10-0 6-5 55 45 35 25 0-15 0075 0 038 0-019

9.25%0 45 55 60 65

+ +

(+) -

Areas examined for IgG staining are those listed in Table 1. (+) Indicates a weakly positive reaction.

D ISCUSSION

The results of this study indicate that apparently specific immunofluorescence reactions for IgG and IgA can be induced in renal basement membrane and reticulin by washing unfixed cryostat sections in PBS before staining with the appropriate conjugate (Table 1). It seems unlikely that these immunofluorescence reactions represent serum proteins passively adsorbed to connective tissue structures. If this was so, we would expect a similar distribution of IgG and IgA, but this was not the case. The anti-IgA conjugate reacted with peritubular reticulin and not with tubule basement membrane as defined by Scott (1957; Scott & Rowell, 1967). The 'dot-dash' distribution of IgA staining in GBM (Fig. 4) also differed from the smooth linear distribution of IgG (Figs 2 and 3). Finally, IgM was detected in only two renal biopsy specimens, which also argues against passive adsorption of serum proteins. Although the immunofluorescence observed in this study satisfied rigid criteria of specificity, we cannot say whether the staining represents true molecular IgG and IgA or crossreacting antigens. Tan & Kaplan (1963) reported that a serum beta-protein was detected in washed sections of rat kidney, and Milazzo (1959) reported that antisera prepared against purified human renal basement membrane antigens also reacted with serum immunoglobulin. In more recent work it has been shown that anti-IgM sera react with a carbohydrate component in lymphocyte membranes that is antigenically similar to the Fc portion of IgM (Merler, Gatien & DeWilde, 1974) and that because of this cross-reaction some of the IgM fluorescence seen on lymphocytes may be spurious. It is conceivable that an analogous system of cross-reacting antigens mimicking IgG and IgA could be responsible for some of our results.

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A charge-effect is suggested by the observation that staining is obtained by washing in ionized but not in non-ionized solutions (Table 2), and this could be responsible for exposing IgG-like and IgA-like antigens in connective tissues. If conformational changes do occur due to a charge-effect in ionized solutions, such changes are apparently not allowed if the tissues are first fixed in ethanol, formaldehyde, or glutaraldehyde, and, such changes must be relatively stable insofar as fixation after PBS washing does not abolish staining. Finally, blocking substances may be removed from renal tissues by washing in PBS. If the IgG-like and IgA-like material exposed in this way has antibody function, one might argue that the blocking substances were non-avidly bound antigens. However, there is no evidence that this is so, and it strikes us as being rather unlikely. Whatever the explanation of the findings reported here, it is evident that errors in interpretation of immunofluorescence studies may occur when kidney sections are washed in PBS before staining with appropriate conjugates. In practical terms this means that cryostat sections should probably be fixed without washing in PBS or, if washing is considered essential, non-ionized isotonic solutions such as sucrose can be employed in preference to PBS. If this is done, conjugate dilutions should also be made in the same non-ionized isotonic solution and not in PBS. We thank Dr C. Piel, University of California, for the renal biopsy material, and Dr H. Fudenberg for the anti-Ig sera. REFERENCES

FAULK, W.P. & HIJMANS, W. (1972) Recent developments in immunofluorescence. Progr. Allergy, 16, 9. FAULK, W.P. & HOUBA, V. (1973) Immunological reactions with chromic chloride-treated erythrocytes. J. immunol. Meth. 3, 87. FAULK, W.P., MCCORMICK, J.N., GOODMAN, J.R. & PIEL, C. (1974) Glomerular beta-lipoprotein in childhood renal disease. Scand. J. Immunol. 3, 665. HOLBOROW, E.J. (1971) Introductory review: tissue antibodies in experimental systems and in human autoimmunity. Ann. N.Y. Acad. Sci. 177, 171. MCCORMICK, J.N., FAULK, W.P., Fox, H. & FUDENBERG, H.H. (1971) Immunohistological and elution studies of the human placenta. J. exp. Med. 133, 1. MERLER, E., GATIEN, J. & DEWILDE, G. (1974) Significance of immunofluorescent staining of lymphocytes with antisera to IgM immunoglobulins. Nature (Lond.), 251, 652. MILAZZO, S.C. (1957) A study of the immunological properties of reticulin. J. Path. Bact. 73, 527. SCOTT, D.G. (1957) A study of the antigenicity of basement membrane and reticulin. Brit. J. exp. Path. 38, 178.

SCOTT, D.G. & ROWELL, N.R. (1967) Alterations in the antigenic constitution of renal glomerular capillaries accompanying the histological maturation of renal glomeruli in the rat. Ann. rheum. Dis. 26, 10. TAN, E.M. & KAPLAN, M.H. (1963) Immunological relation of basement membrane and a serum betaglobulin in the mouse. Demonstration of renal basement membrane alteration in mice injected with streptolysin S. Immunology, 6, 331. WANG, A.C., WANG, I.Y.F., MCCORMICK, J.N. & FUDENBERG, H.H. (1969) The identity of light chains of monoclonal IgG and monoclonal IgM in one patient. Immunochemistry, 6, 451. WANG, A.C., FAULK, W.P., STUCKEY, M.A. & FUDENBERG, H.H. (1970) Chemical differences of adult, fetal, and hypogammaglobulinemic IgG immunoglobulin. Immunochemistry, 7, 703. WEIR, D.M. (1973) Immunological methods applied to the study of tissue antigens and antibodies. Handbook of Experimental Immunology (ed. by D.M. Weir), 2nd edn, chapter 35.1. Blackwell Scientific Publications, Oxford.

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IgG and IgA antigens in human renal basement membranes.

Clin. exp. Immunol (1975) 21, 75-81. IgG AND IgA ANTIGENS IN HUMAN RENAL BASEMENT MEMBRANES J. N. McCORMICK AND W. PAGE FAULK Rheumatic Diseases Unit...
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