Changes in serum complement and fucose after skin grafting in guinea pigs

CHANGES AFTER EDWARD

D.

IN

SERUM COMPLEMENT SKIN GRAFTING IN

COPPOLA, AND

M.D.,* FRANCIS

THE ROLE OF THE COMPLEMENT (C’) system in immune hemolysis has been well known and widely studied for many years, resulting in the recent discovery that at least nine separate components are needed to carry this reaction to completion in the guinea pig [ 111. Some components seem to function by activating others, some are rendered inactive during C’-fixation, and some seem to be taken up physically by immune complexes [ 171. Decreases in the levels of serum C’ regularly accompany antigen-antibody reactions other than immune hemolysis in certain experimental models [12, 151 and depressions of serum C’ occur in the acute phase of such auto-immune diseases as systemic lupus erythematosus and glomerulonephritis [7]. Similar depressions have been sought in From the Department of Surgery, Hahnemann Medical College, Philadelphia, Pa. *Assistant Professor of Surgery and John and Mary R. Markle Scholar in Academic Medicine. 1Surgical Research Fellow. *Associate in Surgery, Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pa. This work was supported by U.S. Public Health Service Grant AM-07886, American Heart Association Grant 67-858. and National Cancer Institute Grant CA 10287-01. The authors are indebted to Dr. R. A. Nelson, Jr., and Dr. Romeo M. Zarco for assistance and advice, and to Mrs. Judith Rounick and Mr. John P. Waldon, Jr., for technical help. Presented at the First Annual Meeting of the Association for Academic Surgery, Lexington, Ky., November 10-l 1, 1967. 308

CRECORIO E.

ROSATO,

AND GUINEA R.

VILLEGAS,

FUCOSE PIGS M.D.,f

M.D.+

experimental animals or patients rejecting skin or renal allografts, but the results have been inconsistent and seem to reflect variations of species, methodology, and types of rejection. For example, decreases in serum C’ are more readily demonstrated during rejection of xenografts [4] than of allografts. Fluctuations in the level ‘of serum C’ and C’ components during allograft rejection could be regarded as inferential evidence for participation of C’ in the effector mechanism of rejection. Such fluctuations could also be of predictive value in establishing a diagnosis of rejection, depending on their time of onset. Should C’ play a role in effecting allograft rejection, then treatment with C’ inhibitors might offer an approach to abrogation of the rejection reaction as originally proposed by Nelson [lo]. For these reasons we have started to investigate the levels of whole C’ and nine C’ components in the serum of guinea pigs after various types of skin grafting. Our preliminary results have uncovered thus far characteristic patterns of response after autografts and allografts are performed in this model. The ready availability of a method for determining serum protein-bound fucose in this laboratory prompted us to measure the levels of this sugar as well in some of these animals [ 131. The B,, fraction of serum, corresponding to the C& component of complement, contains 0.16% fucose as one of its sugars [9]. We therefore thought it possible

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that fluctuations of protein-bound fucose might parallel or reflect changes in serum C’. MATERIAL

AND

METHODS

Linebred American male albino guinea pigs were used in all experiments. We have previously reported the details of skin grafting techniques, postoperative care, and criteria of rejection [3]. Blood for serological and chemical determinations was drawn by cardiac puncture and the serum separated and stored at -70°C. until tested. Whole C’ activity was measured by microtitration using a hemolytic test system modified from Kabat and Mayer [6]. The sheep erythrocytes were optimally sensitized with purified 7s hemolysin prepared in this laboratory. Titers of the nine C’ components, c;, c:, c:, c.:,, qh’ q,, C3’f, Cs:l, and C3:l, were estimated by a similar procedure using purified components and intermediates supplied by Dr. R. A. Nelson, Jr., and Dr. R. M. Zarco. The component being measured was omitted from the reaction mixture; the serum being tested acted as its limiting source. All titrations were done in duplicate using sera of known titer as controls and the results expressed as reciprocals of the titers. Whole C’ Determinations Twenty-seven autografted cases and 31 allografted cases had serial estimations of whole C’ over a period of two weeks beginning one day before grafting. Twenty autografted animals had whole C’ determinations on days -1, 1, 4, 6, 8, 11, 13, and 15, and seven on days -1, 3, 5, 7, 9, 13, and 15. Twenty-two allografted animals had whole C’ determinations on days -1, 1, 4, 6, 8, 11, 13, and 15 and 9 animals on days -1, 3, 5, 7, 9, 13, and 15. Median values for each day were tabulated for each experimental group and correlated with gross and histological observations of the grafts.

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C’ Component Determinations Three autografted cases and four allografted cases had titration of whole C’ and all nine C’ components on days -1, 1, 4, 6, 8, 11, 13, and 15. These titers were individually graphed on logarithmic paper. Fucose Determinations Seven autografted cases and nine allografted cases having determinations of whole C’ also had serum protein-bound fucose measured on days -1, 1, 3, 5, 7, 9, 13, and 15. The method of Dische and Shettles, as modified by Winzler, was used [ 161. A 0.1 ml. aliquot of serum was placed in a centrifuge tube, 3 ml. of 95% ethanol were added, and the mixture was agitated and then centrifuged at 3000 rpm for 20 minutes. This procedure was repeated. The precipitate thus obtained was suspended in 3 ml. of 0.1 N sodium hydroxide. One ml. aliquots of the alkaline hydrolysate were then transferred to test tubes, to which were added 4.5 ml. of 6:l concentrated sulfuric acid to water mixture. A methylpentose standard (4 ug. of fucose per ml. ) and a blank were agitated for ten seconds, heated for 3 minutes in boiling water, and then cooled in tap water. Duplicate samples were prepared in each instance, and after boiling, 0.1 ml. of cysteine reagent (So/Caqueous cysteine hydrochloride) was added to one of the serum samples and omitted from the other to correct for nonspecific color development. After 60 minutes at room temperature, readings were taken on the Beckman spectrophotometer at 396 and 430 mu with transmission through distilled water taken as the zero point. The measurement at two different wavelengths allowed correction for color developed by other sugars. The fucose content was calculated from the differences in the readings at 396 and 430 mu, subtracting the values without cysteine according to the formula shown below.

(O.D. of A at 396 mu - O.D. of A at 430 mu) - (O.D. of B at 396 rnp - O.D. of B at 430 mu) (O.D. of S at 396 mu - O.D. of S at 430 mu) X 12 = milligrams of fucose per 100 ml. of serum 309

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O.D. = the optical density A = the unknown sample with cysteine B = the unknown sample without cysteine S = the standard 12 = the dilutional factor. Median Survival Times Median survival times in allografted cases were calculated according to the method of Litchfield [81.

Whole C’ Determinations Median levels of whole serum C’ determined serially in the two groups of grafted guinea pigs are given in Table 1. Within each group 75 to 85% of the values were clustered exactly at the median titer for that day. Two patterns of response emerged. One day after grafting, and before circulation between the grafts and their beds was well established, whole C’ levels rose to the same extent in both groups. In the autografted group, without rejection, there was a gradual decline to normal levels by the eleventh day after grafting. In the allografted group, with rejection

Table 1. Median Whole C’ Titers in Two Groups of Guinea Pigs Having Skin Grafts Whole C’ Titers (reciprocal) Day of Experimenta

Autografts (27 cases)

-1

640 960 900 960 800 800 800 770 700 640 640 640

7" 8 9 11 13 15 ~ ~___ -__ aSkin grafting 310

-~ done

on day

2

3

4 DAYS

5

6 AFTER

7

s

s

IO

II

12

I2

I4

I5

DRAFTING

Fig. 1. Serial determinations of whole serum C’ in 4 guinea pigs with allogeneic skin grafts. Whole C’ levels were consistently depressed during rejection,

RESULTS

1 3 4 5

-I 0 I

Allografts (31 cases)

__~ 0.

640 960 640 640 500 500 500 500 500 640 640 640 ___-

and a median survival time of 7.5 +- 0.2 days, a fall to normal levels occurred by the third day after grafting. This was followed by a period of depression of whole C’ below normal, which was sustained during the period of clinical rejection (95% confidence limits: 6.9 to 8.2 days). When the grafts were completely rejected, whole C’ levels returned to normal and remained there during the rest of the experiment. Figure 1 shows the characteristic patterns of whole C’ titers in four allografted animals during the two weeks after grafting. In each case there was a transient postoperative rise followed by a sustained depression during rejection and a return to normal by the eleventh day after grafting. C’ Component Determinations The levels of the nine C’ components in three autografted animals either remained unchanged throughout the experiments or rose transiently during the immediate postoperative period. There was no consistent pattern of depression in these animals. In the allografted rejecting group the levels of C{, C;, and C(, also showed no consistent changes. Figure 2 shows the levels of C(, in the same four allografted animals whose whole C’ levels appear in Figure 1. No changes occurred in the levels of this component. But the levels of the six late-acting C’ components were almost uniformly depressed during rejection. In only three of the

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Fig. 2. Serial determinations of the Cl, component of complement in the serum of 4 guinea pigs with allogeneic skin grafts. The levels of C.: were unchanged during rejection (same 4 animalsas in Fig. 1).

Fig. 3. Serial determinations of the C ’ com:itl ponent of complement in the serum of 4 guinea pigs with allogeneic skin grafts. The levels of C ‘ 3% were markedly depressedduring rejection (compare with Figs. 1 and 2).

24 late-acting component determinations was there no depression during rejection, and each of these three exceptions involved a different component. Definite patterns of depression during rejection were noted in C& C::f, and C::,,; but the most marked and consistent depressions occurred in C ?‘, C:‘,, and Cl<:. The levels of CL in four alldgrafted animals are shown in Figure 3. These curves should be compared with the curves for whole C’ and CT, in the same animals (Figs. land2). -

Fucose Determinations

40

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Suum

hcow

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Figure 4 depicts the patterns of median values of serum protein-bound fucose in 7 autografted and 9 allografted guinea pigs. On the third day after grafting there was a slight rise in the level of fucose in the autografted group but a decrease below the preoperative level in the allografted group. This depression below the baseline occurred in every animal bearing a skin allograft. Thereafter, there was a sustained and marked rise in the level of fucose in the autografted group Median Fucosa

Sorum (mg.96)

1

35-

38

33

26 30 24-

21

2D42 IsI8

I2-

+

14

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0 I

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7

9

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7

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after gmfting Allogrofts

Fig. 4. Patterns of serum protein-bound fucose in 7 guinea pigs with skin autografts and 9 guinea pigs with skin allografts. Note the drop in serum fucose on the third postoperative day in the allografted

group. 311

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to 436% above normal. In contrast, the values in the allografted group never exceeded 102% of the preoperative level. The initial rise in serum fucose on the first day after allografting and a consistent drop on the third day corresponded to a similar rise and fall of whole C’ in the same animals on the same days. DISCUSSION The immediate postoperative rises of serum C’ in both our autografted and allografted animals appear to be a nonspecific response to trauma and inflammation and have been noted by others [ 1, 5, 141. Such initial nonspecific rises may account for the failure to demonstrate subsequent depression of serum C’ activity during allograft rejection in certain models. When depressions of serum C’ activity do occur with allograft rejection, these changes seem to be more strikingly reflected in the levels of C’ components than in the levels of whole C’. For example, Guiney and colleagues found depression of the C[, component of human C’ to be a more sensitive indicator of rejection than depression of whole C’ [5]. In our guinea pigs the levels of the late-acting components were severely depressed, while whole C’ levels were moderately depressed and Ci levels were not at all depressed. It seems reasonable to find such species variability in a kinetic system that involves an intricate cascade reaction whose components, with the exception of one ( Ci,g.“. = C f hn = B are not known globulin), 1C to ;l heterologously interchangeable. The experiments we are reporting here support the view that when depressions of whole C’ occur during allograft rejection, simultaneous depressions in the activity of C’ components are more marked. Depressions of whole serum C’ have not been found during rejection of skin allografts in rats [5, 141. Thus, measurable changes in whole serum C’ or C’ components may or may not be found during allograft rejection depending on the species being investigated and the organ or tissue being rejected. When whole C’ depressions do occur, they may re312

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fleet depressions of some components in one model or of other components in another. The difference already referred to between Ci activity in human recipients of renal allografts and guinea pig recipients of skin allografts illustrates this point. Biphasic serum C’ curves similar to those we observed in guinea pig allograft rejection were found by Carpenter and co-workers in patients rejecting renal allografts [2]. The timing of the changes in their patients was later with respect to clinical rejection, but this could be explained by use of an immunoassay which probably measured complement already bound to antigen-antibody aggregates as well as inactive decay products which were antigenically similar (Br,/Br,). In any case an initial rise above normal levels, followed by a fall below normal, appears to be a pattern of C’ activity associated with some types of allograft rejection. We have shown an initial rise in serum protein-bound fucose followed by a fall on the third day after skin allografting in guinea pigs. Since this pentose is one of the carbohydrate moieties of B,c ( C,‘J , these changes might reflect alterations in the levels of this complement component. These data are correlative and only suggest a role for the C’ system in allograft rejection since variations in C’ activity may result from changes in rates of synthesis or catabolism as well as uptake.

SUMMARY -4 consistent pattern of whole c’ depression has been found in the serum of guinea pigs rejecting skin allografts. This depression reflects primarily depression of the late-acting components in this model and does not occur in autografted animals without rejection. The concentration of serum protein-bound fucose, a sugar moiety of the C.:, component of complement, is also consistently lower in the serum of allografted animals with rejection than in autografted animals without rejection. These data support the concept of a role for serum C’ in the effector mechanism of allo-

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graft rejection and suggest that measurements of whole C’, C’ components, and proteinbound fucose may be useful guides to the diagnosis of rejection.

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REFERENCES 1.

2.

3.

4.

5.

6. 7.

Austen, K. F., and Russell, P. S. Detection of renal allograft rejection in man by demonstration of a reduction in the serum concentration of the second component of complement. Ann. N.Y. Acad. Sci. 129:657, 1966. Carpenter, C. B., Gill, T. J., III, Merrill, J. P., and Dammin, G. Instability of the complement system in patients with renal allografts. Transplantation 5:864, 1967. Coppola, E. D., and Villegas, G. R. Serum complement: Changes after skin grafting in guinea pigs. Proc. Sot. Exp. Biol. Med. 125:1071, 1967. Gewurz, H., Clark, D. S., Cooper, M. D., Varco, R. L., and Good, R. A. Effect of cobra venominduced inhibition of complement activity on allograft and xenograft rejection reactions. Transplantation 5: 1296, 1967. Guiney, E. J., Austen, K. F., and Russell, P. S. Measurement of serum complement during homograft rejection in man and rat. PTOC. Sot. Exp. Biol. Med. 115:1113, 1964. Kabat, E. A., and Mayer, M. M. Experimental ImmunochemCtry. Springfield: Thomas, 1961. Lange, K., Wasserman, E., and Slobody, L. B. The significance of serum complement levels for the diagnosis and prognosis of acute and sub-

10.

11.

12.

13.

14.

15.

16. 17.

AND

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GRAFTING

acute glomerulonephritis and lupus erythematosus disseminatus. Ann. Intern. Med. 53:636, 1960. Litchfield, J. T., Jr. A method for rapid graphic solution of time-percent effect curves. J. Pharmacol. Exp. Ther. 97:399, 1949. Miiller-Eberhard, H. J., Nilsson, U., and Aronsson, T. Isolation and characterization of two B glycoproteins of human serum. J. Exp. Med. 111:201, 1960. Nelson, R. A., Jr. Isolation of complement fractions presents a new approach to immunosuppression. J.A.M.A. 191:30, 1965. Nelson, R. A., Jr., Jensen, J., Gigli, I., and Tamura, N. Methods for the separation, purification and measurement of nine components of hemolytic complement in guinea-pig serum. Immunochemistry 3:111, 1966. Rice, C. E. An investigation of some of the factors determining the decrease in complement activity in anaphylactic shock. J. Immun. 75:85, 1955. Rosato, F. E., Villegas, G. R., Cowan, S., and Coppola, E. D. Serum protein-bound fucose in transplantation. Surgery 62:935, 1967. Rother, K., Rother, U., and Ballantyne, D. L., Jr. Serum complement activity in rat recipients of small and massive skin allografts. Proc. Sot. Exp. Biol. Med. 124:439, 1967. Seltzer, G., Baron, S., and Fusco, J. A method for removing complement in vivo and its rate of return. J. Immun. 69:367, 1952. Winzler, R. J. Methods of Biochemical Analysis. New York: Interscience, 1955. Yachnin, S. Functions and mechanism of action of complement. New Eng. J. Med. 274:140, 1966.

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