EFFECT OF ANTILYMPHOCYTIC ANTIBODY AND ANTIBODY FRAGMENTS ON SKIN-HOMOGRAFT SURVIVAL AND THE BLOOD-LYMPHOCYTE COUNT IN RATS

1126 EFFECT OF ANTILYMPHOCYTIC ANTIBODY AND ANTIBODY FRAGMENTS ON SKIN-HOMOGRAFT SURVIVAL AND THE BLOOD-LYMPHOCYTE COUNT IN RATS N. F. ANDERSON

K.

JAMES

M.B. Edin.

Ph.D. Birm.

LECTURER

SENIOR LECTURER

M. F. A. WOODRUFF M.D., M.S., D.Sc. Melb., F.R.C.S. PROFESSOR OF SURGICAL SCIENCE

From the

Department of Surgical Science, University of

and Medical Research Council Research Group Clinical and Experimental Problems of Transplantation

Edinburgh,

on

Antisera have been raised in a horse and in rabbits to thoracic-duct lymphocytes from hooded rats. These antisera and IgG globulin prepared from them prolonged skin-homograft survival in rats and produced a sustained lymphopenia. On the other hand F(ab’)2 and Fab’ fragments of the horse IgG, given in a dosage equal to the dose of IgG which increased homograft survival by a factor of 2·5, had no effect on homograft survival and produced only a transient lymphopenia. Normal horse and rabbit serum had no striking effect.

Summary Summary

Introduction

SERUM prepared by immunising an animal with xenogeneic lymphoid tissue causes a fall in bloodlymphocyte count when injected into a member of the species which provided the lymphoid tissue (Chew and Lawrence 1937). Woodruff and Anderson (1963, 1964) showed that the survival of homografts may be greatly prolonged in animals given injections of serum prepared in this way; Woodruff (1967) has reviewed the reports which confirm this finding. (By serum we mean not only serum separated in the usual way from clotted blood but also plasma from which the fibrinogen has been removed by heating at 560C for 30 minutes followed by filtration.) We have determined the

extent to

which these

two

properties are exhibited by IgG prepared from antilymphocyte serum (A.L.S.), and by divalent and univalent antibody fragments of the IgG molecule. Materials and Methods Rabbit-anti-rat A.L.s. was prepared as described by Woodruff and Anderson (1964) by immunising rabbits by repeated intraperitoneal injections of rat thoracic-duct lymphocytes. The first three injections were given at weekly intervals; each contained 2 B 108 lymphocytes and the rabbits were bled

10 days after the last injection. The rabbits were bled again on several occasions 10 days after they had received a booster injection of 1 to 2 x 108 lymphocytes. Horse-anti-rat A.L.S. was prepared by immunising a horse with three intravenous injections of 1-07x10’’ to 1.30 x 109 thoracic-duct lymphocytes (> 95% viability) at weekly intervals, followed by a fourth injection 2 weeks later, and bleeding 11 days after the last injection. IgG was prepared from horse-anti-rat A.L.S. by repeated (twice) sodium-sulphate precipitation (final concentration 14% w/v) followed by diethylaminoethyl cellulose batch chromatography on WhatmanDEll’ exchanger. The final product was concentrated by lyophilisation and reconstituted to yield a preparation containing 1 g. protein per 100 ml. This product was shown by immunoelectrophoresis (using a rabbit-antihorse serum) and polyacrylamide-gel electrophoresis to contain only small amounts of contaminating protein, presumably IgA(T)-a protein present in large amounts in hyperimmunised horses and is the main source of commercial antitoxins (see Weir and Porter [1966]). The F(ab’)2 portion of the antibody molecule was prepared by digestion with pepsin in 0-1 M acetate buffer (pH 4-0) at a ratio of 1 part enzyme to 50 parts IgG w/w. Digestion was shown to be complete, by immunodiffusion analysis and by the fact that while the product retained its capacity to agglutinate rat lymphocytes it did not lyse them in the presence of complement. The univalent Fab’ fragment was obtained by reduction of the F(ab/)2 in phosphate buffer (pH 7-2) containing 0-1 M cysteine hydrochloride and subsequent alkylation by dialysis against large volumes of iodoacetamide (0-02 M) in the same buffer. Completeness of reduction was shown by the fact that the product did not agglutinate lymphocytes in vitro (inverse titre, < 8). The preparations of both antibody fragments was concentrated by ultrafiltration to yield a protein content of 1 g. per 100 ml. Crude A.L.S. contained erythrocyte agglutinins in fairly high titre. Before use it was inactivated by heating (56°C for 30 minutes) and absorbed with rat erythrocytes (1 volume erythrocytes for horse-anti-rat and 1/2 volume for rabbit-anti-rat A.L.S.). IgG and antibody fragments did not require absorption. Further details of these procedures, and the lymphocyte agglutinin and cytotoxin titres of the various preparations, are given elsewhere (James and Anderson 1967, Woodruff, James, Anderson, and Reid 1967). Two types of experiment were performed. In the first, rats were given a course of daily intraperitoneal injections of A.L.S., normal horse serum (N.H.s.), normal rabbit serum (N.R.S.), or one of the preparations derived therefrom, and during the course each animal was given a homograft of skin. Total and differential blood-leucocyte counts were performed at least once per week. The injections were given at the same time each day and if a blood-sample was required it was taken just before the injection. In the second type of experiment total and differential blood-leucocyte counts were determined in rats before, 4 hours

TABLE I-EFFECT OF ANTILYMPHOCYTIC ANTIBODY AND ANTIBODY FRAGMENTS ON SURVIVAL OF SKIN HOMOGRAFTS IN RATS

1127 TABLE II-EFFECT ON BLOOD-LYMPHOCYTE COUNT OF A SINGLE INTRAPERITONEAL

INJECTION

OF ANTILYMPHOCYTIC ANTIBODY AND

ANTIBODY FRAGMENTS

after, and

24 hours after A.L.S., N.H.S., N.R.S., or

a

single intraperitoneal injection of of the preparations derived

one

therefrom.

Blood-samples were obtained by amputating the tip of the tail under ether anxsthesia. Skin grafting was performed by the method of Woodruff and Simpson (1955). Results

The survival of skin homografts in treated and control animals is shown in table i. Whole A.L.S., and IgG derived from it, produced the same degree of prolongation of skinhomograft survival. On the other hand, neither divalent nor univalent antibody fragment given in the same dosage (i.e., same weight of fragment and of IgG) produced any significant increase in homograft-survival time. N.H.S. and N.R.S. also had no effect. The fall in the absolute blood-lymphocyte count 4 hours and 24 hours after a single intraperitoneal injection of N.H.S., horse-anti-rabbit A.L.s., and preparations derived therefrom, expressed as a percentage of the pre-injection count, is shown in table 11. Once again A.L.s. and IgG in comparable dosage produced a similar effect, namely a sharp fall in the blood-lymphocyte count which had not TABLE III-EFFECT ON BLOOD-LYMPHOCYTE COUNT OF REPEATED DAILY INTRAPERITONEAL INJECTIONS OF ANTILYMPHOCYTIC ANTIBODY AND ANTIBODY FRAGMENTS

returned to normal after 24 hours. Both divalent and univalent antibody fragment produced a smaller but quite definite fall in the lymphocyte-count 4 hours after injection, but by 24 hours this had returned to or exceeded the pre-injection level. The effect of repeated injections is shown in table ill. Horse-anti-rat A.L.s. and IgG behaved indistinguishably and produced a sustained fall in blood-lymphocyte count; at 7 days, however, this was significantly less than that produced by the rabbit-anti-rat IgG. Rats treated with horse-anti-rat Fab’ showed only a slight fall in lymphocytecount at 7 days and none at 14 days; those which received F(ab’)2 showed no fall even at 7 days.

Discussion

IgG

has

a

similar effect

to A.L.s. on

homograft survival

and on blood-lymphocyte count. The failure of F(ab’)2 and Fab’

prepared from A.L.s. to of prolongation skin-homograft survival when given in a dosage equal to the dose of antilymphocytic IgG which increased homograft survival by a factor of 2-5, like their failure to inhibit humoral antibody production (James and Anderson 1967), would seem to be related to the lack of toxicity of these preparations for lymphocytes. This conclusion is consistent with the observation reported by Woodruff, Reid, and James (1967) that F(ab’)2 from horse-anti-human A.L.s. is as effective as an equal dose of IgG in stimulating uptake of tritiated uridine and tritiated thymidine by human lymphocytes in culture. If, as seems likely, this holds good also in vivo, and in species other than man, it would suggest that the immunosuppressive effect of A.L.S. and IgG is not due simply to sterile activation of lymphocytes, though, as Levey and Medawar (1966) have suggested, this may play some part. One way to obtain further light on the question would be to examine the effect on immunological responsiveness of considerably larger doses of F(ab)2 than those we have used so far, and this we plan to do. The cytotoxic effect of A.L.S. and IgG may be manifested in vivo by a fall in the blood-lymphocyte count. There is cause

evidence, however, that A.L.S. may cause a high degree of immunosuppression, including suppression of homograft rejection, in the absence of striking lymphocytopenia (Woodruif 1967). It seems possible that in this event a subpopulation of sensitised lymphocytes may have been selectively destroyed or inactivated. When there is a striking and persistent fall in the bloodlymphocyte count it seems likely that many lymphocytes are destroyed. Sequestration of lymphocytes may also play a part, however, and may well account for the rather transient lymphocytopenia which follows injection of F(ab’)2 and Fab’, since these agents do not bind complement. It would scarcely be surprising if antibody-coated cells were to become trapped in various parts of the capillary bed or even removed from the circulation. Direct evidence of this is lacking, however, and we are therefore embarking on an autoradiographic study of the distribution of IgG-coated lymphocytes when injected into untreated isogeneic animals, and also of labelled but non-coated lymphocytes injected into isogeneic animals which are subsequently treated with antilymphocytic IgG. Requests for reprints should be addressed to M. F. A. W., Departof Surgical Science, University of Edinburgh Medical School, Teviot Place, Edinburgh 8. ment

1128 VARIABILITY OF GASTRIC SECRETION IN PATIENTS WITH AND WITHOUT THE ZOLLINGER-ELLISON SYNDROME

thus the lack of reliability, of gastric secretory studies when these patients were subjected to repeated testing. Methods

DANIEL H. WINSHIP M.D. Texas ASSISTANT PROFESSOR OF MEDICINE

EDWIN H. ELLISON M.D. Ohio PROFESSOR OF SURGERY

From the Milwaukee

Marquette University School of Medicine and County General Hospital, Milwaukee, Wisconsin, U.S.A.

Data from repeated gastric secretory studies (augmented histamine tests, 12hour overnight tests) in eight patients with peptic ulcer are presented. Four patients had proven Zollinger-Ellison syndrome; in the other four this diagnosis was suspected but could not be confirmed at surgery. In seven patients, striking variations in the secretory patterns were observed on repeated testing. The basal secretion exhibited the widest fluctuations resulting in important alterations in calculated ratios of basal to stimulated secretion. Several of the investigations failed to suggest the correct diagnosis or suggested an erroneous diagnosis, while tests in the same patients performed at different times were often consistent with the correct diagnosis. Caution is therefore warranted in the interpretation of gastric secretory data in any patient in whom the Zollinger-Ellison syndrome is suspected.

Summary

Introduction

THE clinical diagnosis of the Zollinger-Ellison syndrome depends largely on various measurements of gastric acid secretion. Of these, the basal acid output, either in 1 hour (Aoyagi and Summerskill 1966) or after 12 hours overnight (Zollinger and Ellison 1955, Donaldson et al. 1957), and the ratio of basal to stimulated acid output (Marks et al. 1961) have been most widely used. Ruppert and Greenberger (1966) suggested that a more reliable variable for identification of the Zollinger-Ellison syndrome is the ratio of basal to histamine-stimulated acid concentration. In each of five patients with the syndrome, these workers found this ratio to be greater than 0-6. Other measurements of gastric secretion, on the other hand, were often misleading. We

give here the results of careful gastric analyses on selected eight patients. A diagnosis of Zollinger-Ellison syndrome was confirmed at operation in four patients and suspected, but not confirmed at surgery, in the remainder. All had serious, recurrent peptic-ulcer disease and all underwent gastric surgery with the tentative preoperative diagnosis of Zollinger-Ellison syndrome. We have demonstrated the

frequent variability, and

DR. ANDERSON AND OTHERS: REFERENCES

Chew, B., Lawrence, J. S. (1937) J. Immun. 33, 271. James, K., Anderson, N. F. (1967) Nature, Lond. 213, 1195. Levey, R. H., Medawar, P. B. (1966) Proc. natn. Acad. Sci. 56, 1130. Weir, R. C., Porter, R. R. (1966) Biochem. J. 100, 63. Woodruff, M. F. A. (1967) J. clin. Path. (in the press). Anderson, N. F. (1963) Nature, Lond. 200, 702. (1964) Ann. N.Y. Acad. Sci. 120, 119. James, K., Anderson, N. F., Reid, B. L. (1967) in Ciba Fdn Study Group on Antilymphocytic Serum (edited by G. E. W. Wolstenholme and M. O’Connor); p. 58. London. Reid, B. L., James, K. (1967) Unpublished. Simpson, L. O. (1955) Plast. reconstr. Surg. 15, 451. W.

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histamine test (Kay 1953) was performed 12-hour overnight fast, a Levin tube was placed under fluoroscopic control with its tip in the gastric antrum, or, in cases with prior partial gastric resection, the tip of the tube was just at the gastroenterostomy. Gastric contents The patient maintained were continuously hand aspirated. the left lateral decubitus position except to roll frequently to the back and right side to facilitate complete emptying of gastric contents. Saliva was collected in a gauze pad held in the mouth, supplemented by expectoration as needed. After the residual gastric contents were aspirated, measured, and discarded, a 1-hour basal collection was performed. Midway through this period, diphenhydramine (’Benadryl’)’) was administered intramuscularly at a dose of 50 or 100 mg. After the 1-hour basal collection, histamine acid phosphate, 0-04 mg. per kg. body-weight, was administered subcutaneously. Gastric secretions were subsequently collected for four 15minute periods. The volume of each sample was measured, pH was determined with the glass electrode, and acid concentration (titratable acidity) was measured by electrometric titration to pH 7 with 0-1 N sodium hydroxide. The acid output of each period was calculated by multiplying the volume of the sample (in litres) by its acid concentration (in mEq. per litre). The following acid outputs were calculated: (1) basal (1 hour) (B.A.o.), (2) maximal (M.A.o.), total from the posthistamine hour (Marks et al. 1961); and (3) peak (P.A.o.)twice the sum of the two consecutive post-histamine periods of highest acid output (Baron 1963). We also determined three ratios: B.A.O./M.A.O.; B.A.O./P.A.O.; and the ratio of the basal acid concentration to the peak post-histamine concentration (Ruppert and Greenberger 1966). Overnight (12-hour) gastric secretion was collected by intermittent mechanical suction applied to the nasogastric tube between 8 P.M. and 8 A.M. Patency of the tube was checked hourly. The total volume of secretion, pH, and acid concentration were measured as above. At least 2 days elapsed between any two studies. All patients were well hydrated. None had gastric outlet obstruction nor The

as

augmented

follows: after

were

a

they taking ulcerogenic or anticholinergic drugs. Results

Patients with Proven

Zollinger-Ellison Syndrome (table i)

Repeated studies show striking variations in basal acid concentration and output (including nocturnal secretion) in all but one patient (no. 1), in whom the results of two augmented histamine tests varied little. Varying ratios of basal to stimulated acid concentration and output primarily reflect the fluctuations in basal secretion. In patient 3 neither of the augmented histamine tests produced suspicious results. The two nocturnal studies, however, were notably different. Whereas both revealed basal hypersecretion, the second nocturnal study demonstrated acid concentration and output values far in excess of the peak histamine stimulated values. Haemorrhage from a duodenal ulcer requiring emergency surgery precluded repeated augmented histamine tests in patient 4. Comparison of one histamine test with the nocturnal study, however, demonstrates a striking difference in the character of basal secretion in the two studies. Values for P.A.o. and M.A.o. were fairly consistent in repeated studies. All

patients in this

group were treated by total Three had metastatic islet-cell carcinoma, gastrectomy. 3 had microadenomatosis of the pancreatic islets. patient