Immunology of Pyelonephritis III. Effect of Colloidal Carbon

0022-534 7/82/1283-0624$02.00/0 Vol. 128, September

THE JOURNAL OF UROLOGY

Copyright © 1982 by The Williams & Wilkins Co.

Printed in U.S.A.

IMMUNOLOGY OF PYELONEPHRITIS III. Effect of Colloidal Carbon JAMES A. ANGEL,* JAMES A. ROBERTS,t T. WOODIE SMITH, JR.:j:

AND

NICHOLAS R. DI LUZIO

From the Departments of Urology and Physiology, Tulane Medical Center, New Orleans, Louisiana, and the Delta Regional Primate Research Center, Covington, Louisiana

ABSTRACT

We induced macrophage suppression with colloidal carbon in monkeys to evaluate the role of the macrophage in bacterial pyelonephritis. The disease was more severe, with prolonged bacteriuria and renal abscess formation, but no more severe scarring than usual. This appeared to be due to the suppression of phagocytosis that occurred, since though the humoral response was delayed it did occur, and the usual response in regional lymph nodes was found. The histologic appearance of chronic pyelonephritis (CPN) is one of a mononuclear cell infiltrate consisting of macrophages, T lymphocytes and B lymphocytes. 1 For this reason a cellular immune response to persistent bacterial antigen has been suggested as the etiology of CPN. Miller and associates 2• 3 and Coles and associates,4 however, have shown that T lymphocyte deficient animals had no significant difference in the course of pyelonephritis when compared to animals not T lymphocyte deprived. We found in studies in the monkey that pathologic change was slight when bacterial antigen (heat killed bacteria) was inoculated when compared to live bacteria. 5 Bacterial antigen also suppressed the cellular immune response, but the inflammatory response was also decreased though the humoral response was normal. Since T lymphocyte deficiency did not affect pyelonephritis, 3 • 4 we and others feel that the inflammatory response might be the most important factor in renal damage that occurs from bacterial infection. 5• 6 Phagocytosis by macrophages, as well as processing of antigen, has been considered to be the initial common step for mounting both humoral and cellular immune response by the host. 7• 8 Thus, if either the macrophage-mediated inflammatory or immune response is important in the etiology of CPN, macrophage impairment should alter the course of pyelonephritis. We induced macrophage suppression with colloidal carbon in rhesus monkeys to evaluate the role of the macrophage in the immune response in pyelonephritis. METHODS

Adult female rhesus monkeys (Macaca mulatta) 3.5-4.5 kg. were used. They were divided into 2 groups. The 1st group of 6 monkeys received 0.3 gm./kg. colloidal carbon (Pelikan, C 11/ 1431a, Gunther Wagner, Hanover, Germany) intraperitoneally (CBIP) to create reticuloendothelial (RES) impairment. After 48 hours the animals were cystoscoped and a 4F ureteral catheter was passed up l ureter for a distance of 10 cm. E. coli 04, 1 X 109 organisms/ml. were injected through the catheter until pyelotubular backflow (PTB) was observed under fluoroscopy. This method of inoculation has been shown previously to cause acute pyelonephritis consistently. 9 The other group of 10 monkeys received bacterial inoculation only, in the same manner. Accepted for publication January 22, 1982. Supported by USPHS grants 2R01 AM 14681 and 2P40 RR 00164. * Supported by an American Urological Association/National Kidney Foundation Fellowship. t Requests for reprints: Department of Urology, Delta Regional Primate Center, Covington, Louisiana 70433. t Research Trainee, supported by USPHS Research Training Grant AM 07212-02. 624

All of the animals were studied before the inoculation and at weekly intervals afterwards. The studies included complete blood counts, urine culture, intravenous pyelogram, renal scan with 131 I hippuran, lymphoblast transformation, urinary bacteria immunofluorescence, 0 antibody titer, and phagocytic rate and killing as previously described. 5 Briefly, urine was cultured with the use of a calibrated loop and bacteria were typed with direct bacterial agglutination with type specific antiserum. Bacteria found were tested for antibody coating with the immunofluorescence (IF) method of Thomas and associates10 testing for IgG and IgM only. Indirect hemagglutination was used for serum O antibody titers. In vitro phagocytic indices were determined with the method of Cohn and Morse11 with the use of whole blood and incubation with bacteria for 2 hours. For lymphocyte transformation (LT), the mitogens used were phytohemagglutinin (PHA), pokeweed (PM) and O antigen from heat killed bacteria. Lymphocytes were separated from blood or tissue by means of Hypaque-Ficoll and cultured in RPMI (Gibco) with L-glutamine, penicillin at 100 U/ml., streptomycin at 100 µg./ml. and fetal calf serum (15 ml./dl. RPMI). The cells (1 x 105 per well in a microtiter plate) were cultured in duplicate with mitogen for 5 days at 37C under 5 per cent CO 2 and then reincubated for 18 hours after the addition of 3 H thymidine. Cell viability (95 per cent) was determined by trypan blue exclusion at the time of cell harvest. Cultures were deposited on glass fiber paper and washed with PBS by means of an automatic sampler harvester. Incorporated radioactivity was counted in a Beckman LS 150 system and results were expressed as net counts/minute (cpm). (cpm experimental minus cpm non-stimulated cells.) The study was conducted on inguinal nodes and blood prior to the study, on blood at weekly intervals during the study, and on blood, spleen and nodes in the region of the infected kidney at sacrifice. The animals were sacrificed 3 weeks after inoculation of bacteria and the kidneys cultured and studied grossly; tissue was also fixed in 10 per cent buffered formalin for hematoxylin and eosin sections and in glutaraldehyde for transmission electron microscopy. Samples for transmission electron microscopy were placed in Karnovsky's fixative 12 for 1 hour, then diced into 1-mm. cubes and left overnight. They were subsequently washed 6 times in 0.2 M cacodylate buffer at IO-minute intervals and postfixed in 1.0 per cent osmium tetroxide in 0.1 M cacodylate buffer for 1 hour. A 1:50 dilution of LO M calcium chloride was added to the buffers to enhance the preservation of membranes. Dehydration was carried out in increasing concentrations of ethanol, after which the samples were transferred into propylene oxide and embedded in Spurr's low-viscosity epoxy resin. 13 A Sorvall MT-

625

MACROPHAGE SUPPRESSION IN PYELONEPHRITIS 3

White Cell Count (per ml blood) WBC: x10"> 16

14

Inf.

1

12 CB

l/

10

8

I

I

I

I

I

I I

I

I

X / \

\

........... Inf.only

(n=IO)

x--X CB+lnf.

(n=6)

:-. \

\ \

\

~

I

"'

".. _y.. 't'-- --

to appear black. Microscopic and electron microscopic study of the kidneys revealed that the carbon in the kidneys was within both polymorphonuclear cells and macrophages which must have migrated into the infection areas of the kidneys since they were not observed in the uninfected kidney (fig. 2, A and B). We did not observe carbon within circulating cells. The rate of bacterial phagocytosis was significantly suppressed in the colloidal carbon plus infection group as compared with the infection alone group (p <0.01). Phagocytic killing, however, was not suppressed by the colloidal carbon (fig. 3). Antibody titers to O antigen were determined at weekly intervals as a parameter of humoral immune response to infection. The titers were found to be significantly lower in the colloidal carbon plus infection than in the infection only group (fig. 4). Another parameter of the humoral immune response is lymphoblast transformation using O antigen as a mitogen. No significant difference was found between the Oantigen response to B lymphocytes in the blood, spleen, or regional nodes of carbon-treated versus infection-only animals at any time during the study (fig. 5). The study of response to the B lymphocyte TABLE

6

1. Renal weights after infection*

Group

8

0

I 2 3 Weeks After Infection

4

FIG. 1. White blood cell count of monkeys infected with or without colloidal carbon. There is no significant difference in counts at any time.

Infection only Infection after colloidal carbon

Control (gm.)

Infected (gm.)

11.0 ± 0.2 9.3 ± 0.3

9.9 ± 0.5 11.8 ± 0.3

* Wilcoxan matched pairs signed rank test shows that the infected kidney is significantly different (p < 0.01) between the groups. Values are expressed as the mean± SD.

2 ultramicrotome was used to obtain silver to pale gold sections employing a Dupont diamond knife. Contrast of the sectioned samples was enhanced by staining in the dark with Watson's saturated alcoholic uranyl acetate 14 for 30 minutes and poststained in Reynolds' lead citrate 15 for 15 minutes. All procedures were conducted at room temperature except for the curing of Spurr's resin at 70C. The stained sections were examined and photographed in a Siemens Elmiskop 101 electron microscope. Statistical comparisons were done with Wilcoxan matched pairs signed rank test. RESULTS

Inoculation of bacteria into kidneys by retrograde injection of bacteria in contrast (sodium diatrizoate) solution until PTB was seen fluoroscopically, resulted in consistent production of pyelonephritis. Histologic changes at sacrifice after infection were typical and included papillitis, mononuclear cell infiltrate, and tubular atrophy, ectasia and scarring in the areas of PTB seen on roentgenograms. All of the animals receiving colloidal carbon and bacteria or bacteria alone had a leukocytosis and developed pyelonephritis in the inoculated kidney (fig. 1). A significant clifference (p <0.02) in the length of bacteriuria was found between the groups as the average length of bacteriuria for infection only was 15.3 ± 3.4 versus 21 ± 2.2 days for the carbon black (CB) plus infection group. Although there was no other difference in cellular pattern of chronic disease, the severity of infection by histologic criteria was greater with the colloidal carbon-treated infected animals than with infection alone since abscess formation was noted in the former. This was shown by the fact that infection alone led to a 10 per cent decrease in renal weight from tubular loss and scarring as is usual,5 whereas the administration of colloidal carbon prior to infection led to a 20 per cent gain in weight, as abscess as well as scarring was present (table 1). Carbon was easily identified grossly at the time of sacrifice in liver and lymph nodes within the abdominal cavity and in the infected kidneys of animals given colloidal carbon and E. coli infection. The carbon which was present in the kidney corresponded exactly with areas of pyelonephritis, causing the involved areas

FIG. 2. A, histology of monkey given colloidal carbon prior to infection. Tubule in center filled with leukocytes containing colloidal carbon. Reduced from X500. B, macrophages containing dense material in phagosome (P) as well as in cytoplasm thought to be colloidal carbon (C). Reduced from x7,000.

626

ANGEL AND ASSOCIATES

mitogen PWM, however, showed a significant decrease in response to this mitogen (p <0.04) of cells from peripheral blood in animals given colloidal carbon prior to infection (fig. 5, A). Cellular immune response to infection was evaluated by studying T lymphocyte blastogenesis in response to the mitogen PHA. A significant suppression (p <0.05) of the PHA response was seen in blood lymphocytes in colloidal carbon plus infection versus infection alone at 2 weeks after infection (fig. 5, A). However, by the time of sacrifice at 3 weeks after infection, the PHA response of carbon-treated animals was the same in cells from regional nodes or spleen as occurred in animals only infected (fig. 5, Band C). Qualitative analysis of the local immune response using immunofluorescence studies of urinary bacteria during infection revealed an interesting phenomenon. The normal response is one of early bacterial coating of urinary bacteria with IgG followed with subsequent rise in IgM antibody coating. Carbontreated animals had IgG and IgM antibody coating early with a rapid decrease in coating over time (table 2).

Cellular Phagocytic Rate ..--Inf. (n=l2)

%

x- - -x CB+ Inf. (n=6)

40

30

20

10

DISCUSSION

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2

3

4

Weeks After Infection

Serum O Antibody Titer

Phagocytic Killing Rate

Titer

o/o

100

/-----x

90 I

I

I

I

I

Injection of animals with colloidal carbon particles has been shown to be an effective means of immunosuppression. 16 The ingestion of the inert carbon particles apparently suppresses the ability of macrophages to subsequently process antigen. 17' 18 Macrophage processing of antigen is thought to be a common step in initiating both cellular and humoral immune responses. 19 In the present study we attempted to suppress macrophage function by colloidal carbon and evaluate the subsequent course of pyelonephritis. Animals treated with colloidal carbon intraperitoneally developed more severe pyelonephritis than control infected ani-

><-->< CB+ Inf.

- - - Inf.

3000

(n=4)

(n=12)

I

2500

2000

80

p(.02 p(.05

1500

70 1000 /

/

500

0

2

3

/

4

Weeks After Infection FIG. 3. The mean phagocytic rate (per cent cells with bacteria at 120 minutes) is reduced significantly (NS = nonsignificant difference) at 2 weeks after colloidal carbon and infection whereas bacterial killing is not reduced at any time. Thus when phagocytosed, the bacteria are killed at the same rate.

/

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1_

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/ 0 ....__ _ _ _ _ _ _...___ _ _ _ __,_

0

2

4

Weeks After Infect ion FIG. 4. 0 antibody titer is significantly reduced after colloidal carbon.

TABL:E

4r

Time }.fter Inoculation. (weeks)

(n=S)

Group

I

T

PW~s

:t t-

I

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p= .037

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\

),., I

I

\ x----x-__

/

O'--~--N~S_ _ p~.007

,,

Infection only (n = Colloidal carbon + infection (n = 6)

-:p<.001

NS

/'

NS

2

\

I

\

p=.024

\\

/

I \ p(.OOI

I I

I

/

_ _ _ _ jp(.001

o'--~--.:;\c;;-_-_-_-x,'..,,_.OOi_~-~

IOr

NS

51

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0

O

NS

--........_;

I 2 3 We@ks Aft~r lnf@ction

_...,_,_!

4

LYMPHOCYTE TRANSFORMATKJN Spl@@m • Inf. (1)

Carbon blacl<+lnf. (CB)

)!

In= 5l

(n=7) CPM

x10'

NS

p=.037

p=.053

8

6

I

'

:r

-,

--,-2-.

1-

T

PWM

-

Ji,

~

0 )__L____&~

_k____.

PKA

0

L YMPHCCYTE TRANSFORMATION Infected Rr;,gional ~Jod!l = lnUil

xCarbon blnck+lnf. (CB)

(n=7)

ln=5)

8

6

4

IgG

5

2

4

4

2

3

IgM

IgM

4

3

2

2

2

mals. Prolonged bacteriuri.a, and more severe histologic changes with abscess formation, were characteristics of the carbontreated animals with pyelonephritis. The presence of carbon particles within many of the macrophages was verified at sacrifice by light and electron microscopy. Macrophage impairment by the carbon particles was demonstrated by showing a significantly depressed phagocytic rate. In contrast, macrophage functions other than phagocytosis may have been maintained as denoted by the finding that there was no suppression in macrophage killing of bacteria in colloidal carbon-treated animals. Similarly a significant suppression of the humoral immune response was achieved as manifested by reduction of the 0 antibody titer and by a diminished blood and splenic B lymphocyte response to PWM. The adverse effects of inhibition of macrophage function, as reflected by the development of pyelonephritis and bacte:riuria, may reflect, in part, impaired phagocytic function as well as humoral immune response. This may be due to depression of phagocytosis by polymorphonuclea:r cells as well as monocytes. The colloidal carbon used is a nonspecific inhibitor of macrophage function, and might well also interfere with other immune functions. Primary cellular immune response to pyelonephritis in the n.1onkey is mounted by lymphocytes of the nodes in the region of the involved kidney. 5 Although colloidal carbon-treated animals showed a significant suppression of the cellular immune response of blood and splenic lyn1phocytes, no suppression was produced in regional node T lymphocytes, the primary focus of cellular immune response in pyelonephritis. The significance of this in the course of the disease is not fully understood, since both Miller and Coles have shown that nearly complete total ablation of T lymphocytes in rats has not changed the course of pyelonephritis in these animals. 7 · 8 Grover and Loegering 19 have ,,,,:.,.,,nv reported that depression of phagocytic function, by the injection of erythrocyte stroma or foreign particulates such as colloidal carbon or gelatinized. reticuloendotheHal test lipid emulsion, was generaJJy associated. with decreased median survival time or increased QVeJ"all to p-,rnp·nn,Al'l'tCJ nPMtA,nh" in rats. the enhancement of modified a variety of sitic 25 infections. Renal candidiasis was »1i,;11111cm1 treated mice. 26 These composite in conjunction with present observations, denotes the importance of macrophage mediated phagocytic and other immune responses in host resistance to infections. Although the use of colloidal carbon (a nonspecific agent) may be questioned, it appears that a functional macrophage population is of importance in cellular and hurnoral-mediated host defence mechanism against pyelonephritis.

)'.

" 2

IgG

IgG

* In 4 monkeys bacteriuria cleared after 1 week.

NS

PHA

3

2 IgM

10)*

'I. //~.05

3

0

fi?uorescent antibody coating of bacteria in urine

x---xlnf. +cm-bor. blod

REFERENCES

X

X

:, -.-

7

0 PWM

X

': PHA

L Smith, J. W., Adkins, M. J. and McCreary, D.: Local immune response in experimental pyelonephritis in the rabbit. I. Morpho-

0

FIG. 5. Lymphocyte transformation of: (A) blood cells (done

weekly), (B) splenic cells at sacrifice, and (C) cells from regional nodes

from near infected kidney. (NS = nonsignificant difference. Significant p values are seen next to mean value which was compared to control values-bar shows 1 standard deviation of control. P values between lines compare the 2 groups, infection versus infection + colloidal carbon.)

628

2. 3. 4. 5. 6. 7. 8. 9.

10.

11. 12. 13.

ANGEL AND ASSOCIATES

logical and functional features of the lymphocytic infiltrate. Immunology, 29: 1067, 1975. Miller, T. E., Burnham, S. and North, J. D. K.: Immunological enhancement in the pathogenesis of pyelonephritis. Clin. Exp. Immunol., 24: 336, 1976. Miller, T., Burnham, S. and Simpson, G.: Selective deficiency of thymus-derived lymphocytes in experimental pyelonephritis. Kidney Int., 8: 88, 1975. Coles, G. A., Chick, S., Hopkins, M., Ling, R. and Radford, N. J.: The role of the T cell in experimental pyelonephritis. Clin. Exp. Immunol., 16: 629, 1974. Roberts, J. A., Domingue, G. J., Martin, L. N. and Kim, J. C. S.: Immunology of pyelonephritis in the primate model: live versus heat-killed bacteria. Kidney Int., 19: 297, 1981. Glauser, M. P., Lyons, J. M. and Braude, A. I.: Prevention of chronic experimental pyelonephritis by suppression of acute suppuration. J. Clin. Invest., 61: 403, 1978. Nelson, D. W.: Immunobiology of the Macrophage. New York: Academic Press, 1976. Dennert, G. and Lennox, E. S.: Phagocytic cells as effectors in a cell-mediated immunity system. J. Immunol., 111: 1844, 1973. Sabet, T. Y. and Friedman, H.: The effects of RES 'blockade' on antibody formation. IV. Inhibition of plaque-forming cells in spleen cultures treated with carbon particles. Immunology, 19: 843, 1970. Sabet, T., Newlin, C. and Friedman, H.: Effects of RES 'blockage' on antibody-formation. I. Suppressed cellular and humoral haemolysin responses in mice injected with carbon particles. Immunology, 16: 433, 1969. Cohn, Z. A. and Morse, S. I.: Interactions between rabbit polymorphonuclear leukocyte and staphylococci. J. Exp. Med., 110: 419, 1963. Karnovsky, M. J.: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A, 1965. Spurr, A. R.: A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31, 1969.

14. Watson, M. L.: Staining of tissue sections for electron microscopy with heavy metals. II. Application of solutions containing lead and barium. J. Biophys. Biochem. Cytol., 4: 475, 1958. 15. Reynolds, E. S.: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17: 208, 1963. 16. Sabet, T. Y.: The mechanism ofimmunosuppression by RES blockage. J. Reticuloendothel. Soc., 11: 503, 1972. 17. Sabet, T. Y., Yound, I. and Friedman, H.: Antigen recognition and localization in RES blockaded mice: morphologic and immunologic studies. Adv. Exp. Med. Biol., 29: 391, 1973. 18. Sabet, T., Newlin, C. and Friedman, H.: The effect of RES blockade on cellular antibody formation to sheep erythrocytes. Proc. Soc. Exp. Biol. Med., 128: 274, 1968. 19. Grover, G. J. and Loegering, D. J.: Effect of erythrocyte debris on reticuloendothelial function and susceptibility to experimental peritonitis. Proc. Soc. Exp. Biol. Med., 167: 30, 1981. 20. Song, M. and Di Luzio, N. R.: Yeast glucan and immunotherapy of infectious diseases. Lysosomes Biol. Pathol., 6: 533, 1979. 21. Kokoshis, P. L., Williams, D. L., Cook, J. A. and Di Luzio, N. R.: Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan. Science, 199: 1230, 1978. 22. Reynolds, J. A., Kastello, M. D., Harrington, D. G., Crabbs, C. L., Peters, C. J., Jemski, J. V., Scott, G. H. and Di Luzio, N. R.: Glucan-induced enhancement of host resistance to selected infectious diseases. Infect. Immun., 30: 51, 1980. 23. Delville, J. and Jacques, P. J.: Therapeutic effect of intravenously administered yeast glucan in mice locally infected by Mycobacterium leprae. Adv. Exp. Med. Biol., 121A: 245, 1980. 24. Williams, D. L. and Di Luzio, N. R.: Glucan-induced modification of murine viral hepatitis. Science, 208: 67, 1980. 25. Cook, J. A., Holbrook, T. W. and Parker, B. W.: Visceral leishmaniasis in mice: Protective effect of glucan. J. Reticuloendothel. Soc., 27: 567, 1980. 26. Williams, D. L., Cook, J. A., Hoffman, E. 0. and Di Luzio, N. R.: Protective effect of glucan in experimentally induced candidiasis. J. Reticuloendothel. Soc., 23: 479, 1978.