The effect of splenectomy on neonatally thymectomized mice

Life Sciences Yol . 5, pp . 1679-1689, 1966 " Printed in Great Britain.

THE

Pergamon Preae Ltd.

EFFECT OF SPLENECTOMY ON NEONATALLY THYMECTOMIZED MICE P. Demos, Jr. , R Leyten and P. De Sourer

Rega Institute for Medical Research, University of Louvain, Louvain - Belgium. (&eceived 25 June 1966) Neonatally thymectomized mice characteristically show a persistently low peripheral blood-lymphocyte level and a marked depletion of lymphocytes is the lymphoid tiesnes. This depletion is associated with a diminished capacity to produce serum antibody to some antigens and a 2, 3. permanent failure of the capacity to reject foreign tissue grafts l'

Such mice grow quite normally during the first month and then may develop a fatal wasting syndrome characterized clinically by lose of weight, hunched posture, ruffled fur, and diarrhea 4,

5.

The pathogenesis of the wasting disease is not known, but several authors have indicated that the strain of mice significa~ly influences the onset and severity of the .disease in the neonatally thymectomized 7, animals 6, 8.

Earlier experiments performed in our laboratory on neonatally thymectomized mice also indicated that incidence of the wasting disease was related to specific strain differences. NMRI mice, an outbred strain, had a low incidence of wasting (about 15 to 25 yf.) . This syndrome occurred more frequently in (C3H x C57B1)Fl hybrids, but if the number of wasting mice in different groups of these thymectomized Fl hybrids is considered, the incidence of wasting was irregular and varied from 30 to 80 7e, the mean percentage being 60 'f, . In nay case, leucocyte counts in the 169

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peripheral blood and histological examinations of the lymphoid tissues of wasting mice showed a severe lymphopenia, which was not observed in thymectomized mice that survived as clinically healthy animals for several months despite the completeness of the thymectomy, This correlation between lymphocyte depletion and wasting syndrome, as well as the strain differences observed, prompted us to investigate whether, added to the thymectomy, removal . of a second major lymphoid organ such as the spleen would increase and regularize the incidence of wasting. Material_ and Methode Mice .

(C3H z C57B1)F 1 hybrids were used throughout our

ezperime~s, Sur¢ical procedures.

Within 16 hours after birth, thymectomy

was performed under anesthesia produced by cooling 9. Splenectomy was done under light ether anesthesia, A small transverse incision was made through the skin and muscle layers overlying the spleen and the organ was ezcised. HematolopCy.

Mice were bled by puncture in the retro-orbital

venous plexus for total and differential white blood cell counts, Blood smears for differential leucocyte counts were stained with May-GrunwaldGiemsa. Antibody measurements .

Sheep erythrocytes in Alsever'e solution

were used to immunize each mouse. The red cells were first washed three times in saline buffered at pH 7.4 with phósphaté and then made up to a t0 ~, suspension in the same saline . Each mouse was given 0 . 5 ml iatrapeFitoneally. Eight days after injection each mouse was bled from the ret"ro-orbital plexus, and serum antibody titrations were performed at room temperature. The titer was read as the last tube in which

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macroscopically visible agglutination was present. Verification of completeness of thymectomy.

Neanatally

thymectomized mice were regularly sac=ificed and the absence of mediastinal thymic tissue was verified nader the dissecting microscope and by microscopic ezamination of the mediastinal contea2s . Ezperiments and Results In a first ezperime~ 40 mice thymectomized at birth were splenectomized at the age of 3 weeks, and 60 neonatally thymectomised mice were kept as a control group. As shown in Fig. 1, development of wasting was less pronounced in the splenectomized group than in the aoasplenectomized group, At the age of 3 months 25 ~ of the splenectomized group and 60 yG of the noasplenectomized mice showed wasting.

-~ f0' e 60-

______ . cont~ol mic~ ( 601 splsnsctomizsd at 3 vwsks
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FIG. 1. Percentage of wasting in neonatally thymectomíáed mice

and neonatally thymectomized mice spleaectomized at the age of 21 days,

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The fact that instead of increasing, the incidence of wasting appeared to decrease in neonatally thymectomized mice splenectomized when 21 days old, led ns to investigate 'the effect of splenectomy performed at earlier ages. Accordingly, 22 and 27 mice thymectomized at birth were splenectomized respectively at the ages of S and 14 days, and 27 aonsplenectomized mice served as a control group. Fig.2 shows that, at the age of 3 months, 20 ~f, of the mice splenectomized when S days old ezhibited wasting, but the mice splenectomized when 14 days old had nearly the same percentage of wasting as the co~rol group, which reached 40 ~..

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FIG. 2. Percentage of wasting in neonatally thymectomized mice and aeonatally thymectomized mice splenectosaized at the age of 8 and 14 days.

The sigaifica~ decrease in the incidence of wasting is mice splensctomized at the age of 21 days could not be duplicated is the second ezpeüment, in which the mice were splenectomized at the ages of S and

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14 days . The control mice in this experime~ had a lower incidence of wasting than those in the first experiment, and the mice spleaectomized at 14 days, in contrast to those splenectomized at the age of 8 or 21 days, developed as much wasting as the nonspleaectomi :ed mice . The variations in the incidence of wasting, as shown in these experiments, render premature any interpretation of the effect of splenectomy oa neonatally thymectomized mice, and the only possible conclndoa is that spleaectomy did not increase the incidence of wasting. Despite absence of täe spleen, a marked increase of lymphocytosis in the peripheral blood appeared is the thymectomized-splenectami:ed mice. The results of total and differential leucocyte counts is the peripheral blood of aeonatally thymectomized mice spleaectomized at the ages of 8 and 21 days, compared to aoasplenectomised thymectomised and noaoperated F1 mice are shown in Table 1. It must be taken into account that the mice bled for the leucocyte counts showed ao symptom of wasting, hence the nearly normal blood formula obtained in the thymactomized aonspleaectomined group. However, no difference in total white blood cell counts or is leucocyte differentiation was recorded in mice developing wasting and belonging to either the control thymectomised or the thymectomi :ed and splenectomized group. The mice were severely lymphopenic in both groups, and the lymphocyte-polymorph ratio was revers :d. This increased lymphocytosis in the peripheral blood of the thymectomi :ed spleaectomized mice, so far as they remaíaed clinically healthy, persisted daring the whole period of the experiments and yet beyond the age of 6 weeks, the number of circulating lymphocytes in these mice was significantly higher than in the i~act F 1 hybrids. The increased lymphocytosis and the sppare~ly lowered incidence of wasting in the mice thymectomized at birth and splenectomised when 8

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or Z1 days old were not followed by a normal serum a~ibody response to sheep red blood cells. The primary response is these mice, at the age of 3 moths, was markedly impaired in comparison with the response recorded in the thymect omized group (Table 2) . We mnet point out that eplenectomy in itself causes a slight decrease in the primary response to sheep red blood cells, and therefore the marked impairment observed in the thymectomized splenectomized mice should be attributed to the combined effects of the eplenectomy and the immunological deficiency of the circulating lymphocytes. DiscusSion Three well-recognized functions are attributed to the spleen storage of blood, destruction of erythrocytes and other blood cells, and the production of lymphocytes is the white pulp

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The increased lymphocytosis

following eplenectomy could be a consequence of a lack in the storage or in the destruction of the lymphocytes. Whether this destruction takes place by direct lyeis in the spleen or indirectly by conditioning them as to make . the lymphocytes more sensitive to proteolytic enzyme must be clarified 11 Splenectomy in neonatally thymectomízed mice has also been followed by increased lymphocytosis, and therefore it would seem that the production of lymphocytes by the white pulp of the spleen is a minor function of this organ. This lymphocytosis appears paradoxical for mice thymectomized at birth, which are regularly characterized by a lymphopenia in the peripheral blood. It suggests that these mice are capable of some lymphoid development, but that the lack of storage capacity or the decreased destruction of the lymphocytes caused by the eplenectomy results in an accumulation of these cells in the peripheral blood. The exact relevance of the lymphocyte deficit to the wasting syndrome has not yet been determined. It would seem, however, that the

1686

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lymphopenia per se in the peripheral blood ie not a direct cause of this syndrome, since it appears from our ezperiments that aeonatally thymectomized mice, in spite of their normal and even increased lymphocytosis resulting from splenectomy, still may develop wasting. The possibility that those circulating lymphocytes are immunologically deficient has not been investigated and needs further study. We could argue in favor of this hypothesis by citing the impairment in antibody production observed in splenectomized animals having a high number of circulating lymphocytes, although any assessment of the antibody response to sheep red blood cells must take into account that splenectomy by itself reduces, to some degree, .

the response to this antigen 12

The nearly normal blood formula and antibody response to sheep red blood cells recorded in neonatally thymectomized mice at the age of 3 moths could be attributed to incomplete thymectomy or ectopic thymic tissues. Incompletely thymectomised animals were rarely found by autopsy, and the high percentage of wasting observed in our thymectomized mice after treatment with cortisone is doses which failed to produce a fatal disease in normal mice ezcludes the prese.ace of ectopic thymic tissues (unpublished data). Other authors have also reported that thymectomized mice are capable of some lymphoid developmn~

13,

and if the animals

survive for a long enough time, they show a progressive restoration of the immune response

14, 15 .

It may be concluded that splenectomy in mice thymectomized at birth does not increase the incidence of wasting, and that the restoration to a normal peripheral blood lymph formula is not followed by the recovery of the immuaological capacity . Experimerts are is progress to verify further the inflnence of splenectomy on the incidence of postthymectomy wasting syndrome .

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Summary Splenectomy performed in young (C3H z C57B1)F 1 hybrids thymectomized at birth has not modified the irregular incidence of wasting in this strain of mice . Despite the removal of a second major lymphoid organ such as the spleen, the lymphopenia in the peripheral blóod resulting from neonatal ihymectomy, was replaced by an increased lymphocytoeis and in some cases these mice developed less wasting. This increased lymphocytosis is not followed by the recovery of the immunological capacity, since the thymectomized-epleaectomized mice still may develop wasting accompanied by a marked lymphopenia and produce less antibodies against sheep red blood cells in comparison with the thymectomized aoaspleaectomized mice. It is concluded that splenectomy does not increase the incidence of wasting disease resulting from neonatal thymectomy. References 1.

J. F. A. P. MILLER, Lancet, ü , 748 (1961),

2.

J. F, A. P, MILLER, Proc, Roy. Soc, B,

3.

R A. GOOD, A. P. DALMASSO, C. MARTINEZ, O. K, ARCHER, J. C . PIERCE and B. W. PAPERMASTER, J, Ezp, Med. , 116, 773 (1962),

4.

J, F. A. P. MILLER, Brit. Méd. J. , ü, 459 (1963) .

5.

D, M. Y, PARROTT and J. EAST, The Thymus in ImmunobioloRy,

156, 415 (1962),

p. 523, Ed, R A. Good and A. E. Gabrielson, Harper and Row Publ. , New York (1964) .

6.

D. M. Y.PARROTT and J. EAST, Nature , 1~5,

7.

D. M. V. PARROTT, Transvl. Bull. , ~ 102 (1962) .

B.

347 (1962) .

L. W. LAW, T. B. DÜNN, N. TRAININ and R H. LEVEY, The Thymus, p. 105, Ed, V. Defendi and D. Metcalf, The Wietar Institute Prees (1964),

9.

J. EAST and D, M. V, PARROTT, J. Eadocrinol . , 24, 249 (1962) .

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C. H. BEST and H. B. TAYLOR, The physiological basis of medical

practice , 3d Ed. , The Williams and Wilkins Co. , Baltimore (1959) . 11 .

M. A. KFi "SALL aad E. D. CRABB, Lymphocytes aad maat cella, p. 25T, The Williams and Wilkins Co. , Baltimore (1959) .

12.

R W. WISSLER, M. J. ROBSON, F. FITCH, W. NELSON aad L. D. JACOBSON, J. Immunol. , 70, 3T9 (1953).

13.

J. EAST and D. M. V. PARROTT, J. Nat. Cancer Iaat. , 33, 6T3 (1964) .

14.

G. ROGISTER, Compt. Rend. Soc. Bio1 . , 158, 404 (1964) .

15 .

J. H. HUMPHREY, D. M. V. PARROTT aad J. EAST, Immnaology . 7, 419 (1964) .