Permanent Granulocytosis in Mice Due to Cortisone

By DAVID R. WEIE AND VIVIAN BRENNER

T HE EFFECT of adrenal cortical hormone and adrenocorticotropic hormone on white blood elements in mice and other mammals is well known,’ and has been partially reviewed by Gordon.8 Lymphopenia, lysis of lymphoid tissue, eosinopenia, granulocytosis and myeloid metaplasia occur in

various combinations, the particular configuration depending upon the animal Downloaded from http://ashpublications.org/blood/article-pdf/14/11/1187/561743/1187.pdf by guest on 30 September 2021 Species. In mice, granulocytosis and myeloid mnetaplasia are particularly striking (luring hormone therapy.7 This led us to test the effect of long-term, intermittent cortisone therapy on mice, in the hope that permanent and irreversible changes might occur. Our preliminary experiments indicated that the granulocytosis might become permanent and not dependent upon continuing cortisone therapy. The experiments reported here confirmed this finding. In addition, a few animals developed apparent myeloid leukemia in the spleen and liver.

METHODS

Animals were obtained from a nonleukeniic strain of albino mice which has been main- tamed in our laboratory for seven years. Male and female weanling mnice aged 25 to 35 (lays were used. Animals were kept in separate cages on a diet of commercial dog chow ad libitum. Blood was obtained by clipping the tail V( in. Total white blood cell counts and differential smears were done at 1 to 4 week intervals. Injections of cortisone#{176} and control solution#{176} were made subcutaneously on the neck and upper back. At autopsy, sections of the liver, spleen, marrow, adrenals and lymph nodes were taken and prepared for histologic stu(ly.

RESULTS

Preliminary experiiiient.-In trial observations, 15 mice were given cortisone intermittently for 1 to 2 week periods, the daily dose being 1.0 mg. Apparently this dose was too high since only three animals survived more than six months of this treatment. Of these three, two showed persistent granulocytosis lasting a long as five months after cortisone was discontinued. In a preliminary experiment, the daily dose of cortisone was lowered to 0.5 mg The results are shown in figure 1. Cortisone was injected during 13 periods each averaging seven days, range 5 to 14 days. The rest periods in between averaged 16 days and the total treatment period was 308 days or 44 weeks. Ten of the 15 animals survived longer than the 44 weeks of treatment. Figure 1 shows the expected granulocytosis and lymphopenia and reversal of the granulocyte to lymphocyte ratio occurring during each period of

From Highland View Hospital, University Hospitals, and the l)epartment of Medicine, Western Reserve University, Cleveland, Ohio. Supported in part by a grant from the National \“itamin Foundation. Submitted Nov. 13, 1958; accepted for publication Jan. 26, 1959. #{176}Supplied through the courtesy of Merck & Co.

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Fmo. 1.-’! lie effect of intermittent cortisone treatment on the white 1)100(1 ccli counts of mice.

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FIG. 2.-Permanent granulocytosis produced in mice by 64 weeks of intermittent cortisone treatment.

cortisone treatment. The length of each treatment period was determined by the occurrence of this reversal. As soon as it was established, the rest period was begun. During the rest period in the early stages of the experiment, the granulocyte to lymphocyte ratio and counts reverted to normal, at which time the next period of treatment was begun. After 30 weeks, the mean granulocyte counts did not return to normal during the rest periods. The granulocytosis remained fixed and permanent for an additional 14 weeks of treatment and persisted for 11 more weeks after cortisone was discontinued. Mean lymphocyte counts were at normal levels. Two of the animals, surviving 55 and 57 weeks, showed histologic findings consistent with myeloid leukemia. These findings will be described below. Final experiment.-For the final experiment, a group of 25 experimental and a group of 20 control weanling mice were used. Instead of being given cortisone the control animals were injected with identical amounts of the sus- pending vehicle for the cortisone. The injection schedules were the same for the two groups, the daily injection volume for each animal was 0.1 ml. and the daily dose of cortisone was 0.5 mg. for the experimental animals. The results are shown in figure 2. The counts during the treatment period Pl,;1u\IANI,;Nl (ilIANULOCYlOSIS IN \l l( E I)U1: fl) (:OIITISONE 1189 Downloaded from http://ashpublications.org/blood/article-pdf/14/11/1187/561743/1187.pdf by guest on 30 September 2021

Fle. 3.-Left: Normal spleen. Rig/it: Spleen of cortisone-treated animal. Note obliteration of architecture and uniform large cell infiltrate. ale not shown since, for the experimental group, they were entirely comparable to those in figure 1 with the expected granulocytosis and! lymphopenia occurring during each treatment period and reversal to normal during the rest

Period. Lymphocyte and granulocyte counts of the control animals remained normal (luring the treatment period. Treatment was gi%’en during 14 periods each averaging 10 (lays, range 6 to 15 days. The rest periods in between averaged 21.5 clays and the total period of treatment was 413 days or 59 weeks. In figure 2 the granulocyte counts are shown for all animals surviving more than 64 weeks; 9 of 25 experimental animals and 11 of 20 control animals. The causes of death of animals (luring the treatment period are not known. hi ()r(ler to minimize the effect of intercurrent infection, counts made within two weeks of death are not charted. Lymphocyte counts during the post- treatment Period were within normal limits for both the experimental and control animals and therefore are not charted. For the experimental animals as compared to the controls there is persisting granulocvtosis which is p1r- ticularly evident between the 73rd! and 93rd! weeks. Morphologic findings-Immature cells (lid! not appear in the peripheral 1)100(1 in significant numbers. Even with the very high counts few cells could be classified as myelocytes or blastic cells. As noted above two of the mice from the preliminary experiment showed morphologic evidence of leukemia. The livers and spleens were enlarged to two or three times normal size. Microscopically the spleen (see figure 3) showed destruction of architecture and diffuse infiltration with polymorphonuclear cells and medium sized mononuclear cells showing many mitoses. Megakaryocytes and nucleated red elements, which normally are present in the mouse spleen, were still present. The liver sinusoids (see figure 4) were diffusely infiltrated with mononuclear cells and mitoses in a distribution similar to that seen in human chronic myeloid leukemia. In one of these two animals this myelocytic type of infiltration involved the adrenals and lymph nodes. 1190 DAVID R. WEIR AND VIVIAN BBENNEB Downloaded from http://ashpublications.org/blood/article-pdf/14/11/1187/561743/1187.pdf by guest on 30 September 2021

Fic. 4.-Left: Normal liver. Right: Liver of cortisone-treated animal. Note diffuse cellular infiltrate between cords of hepatic cells.

Interpretation of the microscopic appearance of the marrow in these animals was difficult. There seemed to be an increase of granulocytic elements, but we could not be sure of this. Mouse marrow normally does not contain fat which is replaced when hyperplasia occurs. As in the spleen the other marrow elements were still present. Differential counts were not done. One other animal from the preliminary experiment and! three from the final experiment showed rather sparse to moderate infiltration of the livers with polymorphonuclear and mononuclear cells without involvement of the spleen. Unfortunately the histologic data are incomplete. Postmortem degeneration occurs very rapidly in mice so that sections from animals not autopsied within a short time after death were usually unsatisfactory for study. Twenty-nine animals from the preliminary experiment and from the experimental group of the final experiment survived more than 44 weks. Satisfactory histologic ma- terial was obtained for only 18 of these 29 animals. For the control animals of the final experiment satisfactory histologic ma- terial was obtained for 7 of 17 surviving more than 44 weeks. None of these showed any infiltrate of the myelocytic type. Although we have not systematic- ally looked for spontaneous leukemia in the old mice of our colony, we have never observed it during the seven years that the colony has been maintained in our laboratory. Recently granulocyte counts and histologic studies were done on 31 mice aged 10 to 23 months. None of these showed evidence of leukemia. Histologic sections of many hundreds of these mice younger than seven months have never shown leukemia. With the data at hand we cannot say that the occurrence of apparent leukemia in the organs of two animals and of leukemoid changes in the organs of four others is of statistical significance. The positive histologic findings did not necessarily occur in animals having the highest granulocyte counts.

DIscusSIoN

In the absence of bacterial infection or necrosis, temporary granulocytosis with or without lymphopenia occurs in mammals under a variety of circum- PERMANENT CRANULOCYTOSIS IN MICE DUE TO CORTISONE 1191

stances, including treatment with various hormones,’7 in vitamin deficiency states,9’#{176} and after stress due to muscular exercise, exposure to physical agents OI to injection of nonspecific substances such as formaldehyde. In all pre- vious experimental situations the granulocytosis has disappeared when the causative agent has been removed. To our knowledge x-irradiation and exposure to benzol are the only other known agents that appear to play a part in the development of permanent and irreversible granulocytosis. The incidence of myeloid leukemia is increased! in chronic benzol poisoning12 and in persons having long-term or very heavy exposure to radiation.1317 There is some clinical evidence1819 that prolonged emotional stress precedes the onset Downloaded from http://ashpublications.org/blood/article-pdf/14/11/1187/561743/1187.pdf by guest on 30 September 2021 of chronic myeloid leukemia in more patients than would be expected by chance. This leads to the tempting hypothesis that prolonged anxiety may have something to do with permanent alteration in granulocyte production. At present there is no good explanation of the mechanisms involved in the production of either the temporary granulocytosis occurring under the various circumstances described above or of the permanent granulocytosis occurring under our experimental conditions. Gordon8 in his review of the effects of hormones has speculated about alteration in the marrow release mechanism hut could find no experimental work on this point in the literature. Valentine20 has reviewed the biochemistry and enzymatic activities of leukocytes in health and disease. He describes in detail the known biochemical activities of leukocytes but can in no way correlate these to provide an explanation for granulocyte overproduction. Likewise no explanation was offered at an international symposium on the leukemias.21 In view of the well known effect of cortisone in decreasing resistance to infection the possibility that the granulocytosis is clue to this factor should be considered. There are several points against this. During the treatment stage, particularly in the early weeks, both the rise and fall of granulocytes is so rapid that it can hardily be expected to be due to infection, and no other investigators have found infection to be a factor in experiments conducted over a very brief term. Second, the persistence of the granulocytosis in the final experiment for as long as 29 weeks after cortisone treatment was discontinued would seem inconsistent with infection due to lowered resistance from the cortisone. Third, in the 18 animals from both experiments that survived more than 44 weeks and were autopsied, there was no gross or microscopic evidence of pyogenic infections. The lungs were not examined microscopically. Since we6 have found the hematologic effect of cortisone and vitamin B6 deficiency to be entirely similar, we have previously speculated about the possibility that cortisone has some effect on the enzymatic activity of the vitamin B group. Recent observations2223 have shown that cortisone in- creases the activity in the liver of transaminase, a B6 enzyme. However, Valentine,20 who studied the transaminase content of white blood cells, could not find evidence that glutamic-aspartic transaminase activity varied consistently in myeloicl leukemia or polycythemia vera. We have attempted to produce permanent granulocytosis l)y inducing intermittent B15 deficiency using desoxypyridoxine on the same experimental plan as described for cortisone in this paper. The experiment was unsuccessful. Only three of 15 1192 DAVID B. WEIR AND VIVIAN BBENNEII mice survived beyond 10 months and permanent granulocytosis was not established, even though intermittent deficiency was induced for 15 months. Our results show that intermittent long-term cortisone therapy produces permanent change in the metabolic mechanism which controls the production of granulocytes or their release from the bone marrow. We may speculate that the change may be connected with alteration of enzymatic activity in which vitamin B6 participates.

SUMMARY

1. Intermittent cortisone therapy given over a period of 59 weeks caused Downloaded from http://ashpublications.org/blood/article-pdf/14/11/1187/561743/1187.pdf by guest on 30 September 2021 permanent granulocytosis in mice from a nonleukemic Albino strain. The granulocytosis persisted for the lifetime of the animals, the longest survival being 97 weeks. Two of 40 animals developed! histologic findings entirely comparable to human myeloid leukemia. 2. It is assumed that such cortisone therapy produces a permanent change in the basic mechanisms which control the production of granulocvtes or their release from the bone marrow. Possibly the metabolic change may be related to inhibition of vitamin B6 activity’.

SUMMABIO IN INTERLINCUA

1. Intermittente cursos de cortisona, administrate dlurante un periodo dc 59 septimanas, causava granulocytosis permanente in muses dc un nonleu- cemic racia albin. Le granulocytosis persisteva durante le complete periodo vital del animales. Le longevitate maximal esseva 97 septimanas. Duo inter 40 animales disveloppava aspectos histologic completemente comparahile a leucemia myeloide in humanos. 2. Es supponite que un tal therapia a cortisona prodluce un alteration permanente in le mechanismos fundamental que governa Ic production dc granulocytos o Ic mechanismo de br liberation ab Ic medulla ossee. Il es possibile que le alteration metabolic es relationate a un inhibition del activitate de vitamina B1;.

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