Lipid A induction of cytotoxic antibody to cultured syngeneic rat kidney tubular cells

Kidney International, Vol. 19 (1981), pp. 275 -280

Lipid A induction of cytotoxic antibody to cultured syngeneic rat kidney tubular cells GEORGE P. HEMSTREET, PATRICIA G. ENOCH, PHILIP R. FINE, and ROBERT WHEAT Departments of Rehabilitation Medicine, Spain Rehabilitation Center, and Microbiology and Surgery, University of Alabama in Birmingham Medical Center, Birmingham, Alabama, and Department of Microbiology, Duke University Medical Center, Durham, North Carolina

dans ces experiences fournit un modèle in vitro qui permet

Lipid A induction of cytotoxic antibody to cultured syngeneic rat

l'étude de l'importance du système immun dans Ia pathogénie de Ia pyélonéphrite.

kidney tubular cells. Syngeneic Fischer rat kidneys repeatedly injected with lipid A induced a cytotoxic antibody to cultured syngeneic rat kidney tubular cells. To test for antibodies in the serum of immunized animals, we disaggregated syngeneic rat kidney tubular cells with collagenase and trypsin and established them in short-term culture. Cultured kidney tubule cells were then radiolabeled 24 hours later with chromium 51 and cultured for an additional 24 hours. Rabbit antirat kidney tubule cell antibody served as the positive control antisera in a complementdependent antibody cytotoxicity assay. Serum samples from animals whose kidneys were innoculated with Re glycolipid were then tested for antibody cytotoxicity. Autoantibody to syngeneic cWtured kidney tubule cells was present in the serum from these animals (P <0.01) as well as in the serum of animals injected i.p. with Re glycolipid when compared with saline controls. The cytotoxic antibody could be removed by absorbing with syngeneic cultured kidney tubule cell membranes. These results suggest that the glycolipid from the mutant strain Re 595 of Salmonella minnesota stimulates a crossreactive antibody to cultured rat kidney tubular cells. The methodology used in these experi-

Induction par le lipide A d'un anticorps cytotoxique contre les cellules tubulaires rénales en culture. Les reins de rats syngéniques Fischer injectés avec du lipide A induisent un anticorps

The mechanism of kidney tubular cell death in acute and chronic pyelonephritis is not completely understood, yet there is presently increasing evidence to implicate the importance of the immune system in the pathogenesis of this disease. Injection of heat-killed Escherichia coli into the kidney parenchyma results in pathologic lesions simulating pyelonephritis, whereas less severe lesions are seen in immunosuppressed animals [1]. Some investigators indicate that bacterial antigens may persist in the kidneys for prolonged periods following active infection [2—4]. Other studies are, however, at variance with these findings [5]. In addition to these reports, Hemstreet has observed that plasma membranes from pyelonephritic kidneys stimulated blast transformation of autologous blood lymphocytes [6]. In contrast, plasma membranes from normal kidneys failed to stimulate the blast transformation.

cytotoxique contre les cellules tubulaires cultivées de rats syngé-

These observations support the possibility that

ments provides an in vitro model for investigating the importance of the immune system in the pathogenesis of pyelonephritis.

niques. Pour évaluer les anticorps dans le serum des animaux immunisés, des cellules tubulaires rénales de rats syngéniques ont Cté désagrégées par la collagénase et Ia trypsine et placées en culture a court terme. Les cellules cultivées ont été marquees

tubular cell membranes may contain bacterial antigens capable of eliciting either a cellular or a humoral autoimmune response. The ability of the endotoxic glycolipid from the mutant strain Re 595 of

24 heures après par chromium 51 et cultivées pendant encore 24 heures. De l'anticorps de lapin anticellules tubulaires rénales de

rat a servi d'antisérum de contrôle positif dans un test

Salmonella minnesota (Re glycolipid) to bind to

d'anticorps cytotoxique dépendant du complement. Les serums des animaux dont les reins avaient été inoculés avec du glycolipide Re ont alors été testes pour la cytotoxicité par anticorps. Des auto-anticorps dirigés contre les cellules tubulaires rénales syngéniques en culture existaient dans le serum de ces animaux (P < 0,01) de méme que dans le serum d'animaux injectés par

plasma membranes suggests that the Re glycolipid may function as an immunologic adjuvant, stimulate crossreactive antibodies, or produce an autoimmune response by acting as a neoantigen [7—10].

voie intra-péritonéale avec le glycolipide Re, cela par cornparaison avec des contrôles qui avaient recu un solute sale. Les anticorps cytotoxiques ont pu être prélevés par absorption au moyen de membranes de cellules tubulaires rénales syngéniques en culture. Ces résultats suggèrent que le glycolipide Re de Salmonella minnesota Re 595 stimule un anticorps croisé contre les cellules tubulaires de rat en culture. La méthodologie employee

Received for publication June 6, 1978 and in revised form April 29, 1980 0085-2538/81/0019—0275 $01.20

© 1981 by the International Society of Nephrology 275

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Herein, we report the ability of endotoxic lipopolysaccharide from Salmonella minnesota Re 595 to stimulate an autoantibody to kidney tubular cells. Salmonella minnesota Re 595, often referred to as a KDO-lipid A derivative [9, 11], was chosen because the lipid A lacks 0 and K antigen specificity [9]. In these experiments, rat kidneys were repeatedly injected with the Re glycolipid. Repeated injection of the Re glycolipid into the kidney stimulated a cyto-

toxic antibody to radioisotope-labeled cultured syngeneic tubular cells. The potential importance of these antibodies in the pathogeneis of pyelonephritis is discussed. Methods

Male and female rats (Fischer, Charles River Breeding, North Wilmington, Maryland), each weighing between 220 and 335 g, were used in these experiments. Throughout the experiments, the rats

had continuous access to Purina rat pellets and drinking water. Endotoxin. A lipopolysaccharide (LPS) known as

Re glycolipid was extracted from a mutant strain (Re 595) of Salmonella minnesota with chloroform and methanol (4:1) and partially purified by meth-

anol precipitation according to the procedure of Chen et al [11].

Immunization technique. To obtain a positivecontrol antibody, three normal rabbits were inject-

ed with rat kidney tubular cells according to the procedure of Linder [12]. The rabbit antirat kidney antibody titer was evaluated by Trypan blue dye exclusion and by chromium 51 isotope techniques [13, 14].

Rats were then immunized with Re glycolipid

(LPS). Under barbital anesthesia, a midline abdom-

weekly for 6 weeks with 1 ml of the Re glycolipid (LPS) at a concentration of 1 mg/ml. A fourth group of six rats were sham injected i.p. with an equal volume of saline. Serum samples were collected from these two groups in the usual manner and tested for cytotoxicity.

Chromium 51 labeling of cultured rat kidney

tubular cells. Prior to chromium 51 labeling, the rat kidney tubular cells were disassociated for culture according to the technique described by Kreisberg, Pitts, and Pretlow [16]. Disaggregated cells (10 X 106) in tissue culture flasks were washed with phosphate buffered saline (PBS) (Gibco). RPMI-1640 medium (3 ml) containing 100 Ci of chromium 51

was then added to each flask. These flasks were then incubated at 370 C for 1 hour. After incubation, the kidney tubular cells were washed with PBS and

removed from the flasks by using 0.25% trypsin. Approximately 3 X 10 cells in 0.2 ml of RPMI-1640 medium with 10% fetal bovine serum (FBS) were placed into each well of a multiwell culture plate (Linbro Scientific, Inc., Camden, Connecticut). The plates were placed in a 37° C carbon dioxide incubator. Antibody-mediated cytotoxicity test. Twenty to twenty-four hours following chromium 51 labeling,

the cytotoxicity test was initiated. After the cultured kidney cells were washed with PBS, 50 l of either antibody or FBS was added to the appropriate wells and allowed to incubate at room temperature for 20 mm. Following the incubation, 200 l of barbital buffer solution was added to each well and incubated for 10 mm. The supernatants were aspi-

rated, and either 50 l of guinea pig complement diluted 1:2 or 50 .il of FBS was added to appropriate

wells and incubated for 45 mm at 37° C. After incubation, 150 jsl of PBS was added to each well.

inal incision was made and the right kidney ex-

From each well, 100 p1 of supernatant was re-

posed. With a 27-gauge one-half-inch needle and a 1-cc tuberculin syringe, 0.05 ml of 100 g/ml concentration of Re glycolipid in normal saline was injected into the lower pole of the right kidney. The contralateral kidney was similarly injected with 0.05 ml of normal saline, and the midline incision was closed with cuticular sutures. The procedure was repeated once weekly up to 7 weeks. One week following the final injection, serum was collected from blood samples obtained via cardiac puncture, and

moved, placed in appropriate tubes, and counted in a gamma counter (Beckman Gamma 8000). The re-

the kidneys were removed for histologic studies

Standardized rabbit antirat chromium 51 assay. A positive complement-dependent antibody cytotoxicity assay was developed. Rabbits were hyperimmunized with rat kidney tubular antigen, and an effective guinea pig complement titer was estab-

[15]. A second group of six rats received intrarenal injections of saline only, and serum samples were collected from them and tested. A third group of six rats were injected i.p. once

maining supernatant and cells were frozen and thawed three times before being placed in the appropriate tubes and counted.

Statistical analysis. Statistical analysis of the data was done by the method of multiple linear regression applied to analysis of variance [17]. Results

277

Lipid A induction of antibody to kidney cells 100

• Normal rats.

90

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80

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1. Plot of the rabbit positive-control serum. The rabbit was immunized four times to stimulate antibody production, and its serum was absorbed with rat liver membranes and rat red blood cells. The complement used in this experiment was at a 1:2 dilution. Fig.

5

— 1:1

lished. Figure 1 demonstrates the cytotoxicity curve of positive-control antisera. Incubation of the kidney tubular cells for 24 hours after chromium 51

labeling reduced the background release to 15 to 20%, as demonstrated at the dilution of 1:1024. There was a slight prozone effect at the higher anti-

body concentration. In subsequent experiments, the positive-control antisera was used at a dilution of 1:16. Cytotoxic antibody to cultured syngeneic kidney

tubular cells by intraparenchymal injection of Re glycolipid. Six animals were injected intrarenally for 6 weeks and the sera tested for cytotoxic antibody to chromium-5 1-labeled cultured kidney tubu-

lar cells. Figure 2 depicts the cytotoxicity titer of six different rat sera after six injections with endotoxic glycolipid from Salmonella minnesota Re 595. These were compared with the serum samples from six intrarenally saline-injected rats and 6 uninjected rats. Maximal killing was observed by using full strength antisera with a 1:2 dilution

of guinea pig complement. Cytotoxicity in the

endotoxin-injected animals was 10 to 13% higher than that in the saline-injected animals. These differences were statistically significant (P < 0.01). Differences in cytotoxicity between the saline-injected and the sham-injected animals were not statistically significant (P > 0.05). Absorption of cytotoxic antibody with kidney tubular plasma membranes. Immune sera were absorbed to plasma membranes prepared from kidney tubular cells by sucrose gradient ultracentrifugation [18]. By an analysis of variance (P <0.05) in three different experiments with antisera obtained from

1:2 1:4 Serum dilution

1:8

Fig. 2. Serum samples from normal and saline-injected rats compared with serum samples from rats injected with Re glycolipid (LPS). Solid triangles (A-A) denote mean data from six rats in-

jected six times with LPS into the right kidney. Open circles (0-0) denote mean data from six rats injected six times with saline into the right kidney. Closed circles (•-S) denote mean data from six normal noninjected animals. At a dilution of 1:1 and 1:2, the serum from the control group and the serum from saline-injected group are not different from each other (P> 0.05)

but are significantly different from LPS-injected animals (P < 0.01). At the 1:4 and 1:8 dilutions, there are no significant differences among any of the groups (P> 0.05).

four different animals, a significant decrease in the

cytotoxic antibody titer in the absorbed antisera was seen when compared with the unabsorbed antisera (Fig. 3). Intraperitoneal injection of endotoxin from Sal-

monella minnesota Re 595 to stimulate cytotoxic antibody. The serum samples from the six animals that were injected i.p. with Re glycolipid were compared with the serum from the positive-control im-

mune rabbits and the six control saline-injected rats. All three groups were significantly different from each other (P <0.01), as shown in Fig. 4. Discussion

A variety of animal models have been established

for investigating the interaction of the host's immune system with an infecting bacterial agent. These models include antegrade and retrograde infection with viable organisms [4, 19], as well as the direct injection of living or dead organisms into the kidney parenchyma [1, 20]. In our studies, we directly injected a defined Re glycolipid from Salmonella minnesota mutant strain Re 595 into the kid-

Hemstreet et a!

278

80

.—. Unabsorbed serum o—o Absorbed serum

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30

20

20

10

1:1 1:2 1:4

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1:1

a, a,

1:2

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0 a, 0

Fig. 4. Plot of percent cytotoxicity vs serum dilution of normal

a,

1:1 1:21:4 Serum dilution

Fig. 3. Results of three separate experiments where serum was taken from animals injected intrarenally with LPS, was absorbed to rat kidney membranes, and then was tested for cytotoxicity against radiolabeled cultured rat kidney tubule cells. A and B

Data of one rat serum. A significant difference between the unabsorbed and the absorbed serum exists only at a 1:1 dilution (P < 0.01). C Pooled serum from two different animals. Again only at the 1:1 dilution of the sera was a significant difference found (P < 0.01).

ney parenchyma. Repeated injection of Re glycolipid into the kidney parenchyma caused a pathologic

lesion similar to that found in pyelonephritis [21, 22]. The value of the experimental design in this study is that (1) it provides a model for studying immunologic mechanisms involved in the pathogenesis of kidney tubular cell death and (2) it provides an

opportunity to investigate the importance of biochemically defined bacterial antigenic components.

A number of immunologic mechanisms may be postulated to explain the development of cytotoxic

antibody in this study and the importance of this development in the pathogenesis of kidney tubular cell death. Re glycolipid (endotoxin) could (1) function as an immunologic adjuvant in breaking self tolerance, (2) stimulate antibodies crossreactive with kidney tubular cells, or (3) bind to cultured kidney tubular cells and act as a neoantigen. It is also pos-

sible that the mechanisms of kidney tubular cell

rat serum as compared with rabbit antirat serum and with serum from rats injected six times i.p. with a 1-mg/mi solution of LPS. Data points are plotted with the standard error of the mean. The data from serum samples were significantly different from each

other at all titers (P < 0.01).

death in vivo may vary depending on the bacterial antigens and may involve none, any one, or a combination of the above. Initially, it was suspected that the endotoxic glycolipid preparation was acting as an immunologic adjuvant stimulating an antibody to kidney tubular cells. This mechanism would be analogous to the antibodies observed in the Heymann nephritis model [23]. In the experiments of Heymann et al, rats produced autoantibodies to several renal tubular antigens when hyperimmunized with kidney cortex and complete Freund's adjuvant. In these studies, the primary antigen was located on the brush border of the proximal kidney tubular cell, and fluorescent antibodies were crossreactive with proximal kidney tubular antigens in other species [24]. In contrast, in our studies, cytotoxic antibodies developed in animals injected i.p., supporting the hypothesis that there are antigenic determinants on the Re glycolipid that stimulate antibodies crossreactive with cultured rat kidney tubular cells rather than the supposition that the endotoxic glycolipid is

acting as an adjuvant. Crossreactive antibodies have been identified in a number of different systems. Antibodies to Tamm-Horsfall protein have been identified by Hanson, Fasth, and Jodal [25]. In

279

Lipid A induction of antibody to kidney cells

addition, strains of E. coli with specific 0-somatic antigens stimulate antibodies that are crossreactive with kidney tubular cells [26]. To our knowledge this is the first time a lipid A component has been

veloped cytotoxic antibody, indicating that the Re

shown to stimulate a cytotoxic antibody that is crossreactive with kidney tubular cells. The low

of pyelonephritis in vivo remains to be defined. Based on observations in other studies, we have

antibody titers in our experimental system (even af-

proposed a requirement for a three-component system involving the binding of Re glycolipid to the kidney tubular cell, antibody, and/or cellular immune mechanisms.

ter hyperimmunization) may be because the antibodies are only cytotoxic to a subpopulation of kidney tubular cells. Fluorescent antibody studies are currently in progress to test this possibility, as well

as to determine if these animals develop immune complex disease after intrarenal or i. p. injection [27].

The antibody to Re glycolipid detected in vitro may also function in vivo by lysing Re-glycolipidsensitized targets, that is, the neoantigen mechanism. In support of this hypothesis, Shands dem-

onstrated the binding of LPS to red blood cell stroma [10], and Springer et al subsequently purified lipid A specific receptors on leukocytes, platelets, and red blood cells [8, 28]. Recent experiments demonstrate that responder C3H/HeN mice kidney tubular cell plasma membranes bind more Re glycolipid than do nonresponder (C3H/HeJ) kidneys. Animal kidney tubular responder cells incubated in vitro with Re glycolipid and anti-Re-glycolipid antibody are also lysed in the presence of complement

(Hemstreet et al, submitted for publication). In vivo, the renal lesion is significantly less severe in

the nonresponder when compared with the responder strain [21]. One explanation for the lack of

killing in nonresponder animals may be that the density of the LPS receptors is too low to effect antibody-complement cell lysis.

The addition of 0 antigens to the lipid A backbone may be another important parameter influencing the pathogenesis of kidney tubular cell death be-

glycolipid has an antigenic determinant cross-

reactive with cultured rat kidney tubular cells. The importance of these antibodies in the pathogenesis

Acknowledgments

Financial support was provided by NIH Young Investigator's Award #CA 24875-02, ACS Fellowship Award #JFCF 398 B, and Spain Rehabilitation Center Grant #547689. Ms. D. Tate prepared the typescript. Reprint requests to Dr. G. P. Hemstreet, III, Urology Division, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA

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