1,25-DIHYDROXYVITAMIN D3 PROLONGS GRAFT SURVIVAL WITHOUT... : Transplantation (original) (raw)
* Abbreviations: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; CsA, cyclosporine; HSV, herpes simplex virus.
1,25-dihydroxyvitamin D3 [1,25-(OH)2D3*] treatments have been tested by others for their ability to prolong experimental cardiac graft survival (1,2). Lemire et al. (2) used the murine cardiac graft model and showed that 1,25-(OH)2D3 had no effect on the prolongation of graft survival. An analog, 1,25-dihydroxy-Δ16-D3 prolonged survival to 27 days. In a review, Bouillon et al (1) cite one experiment in the rat in which cardiac grafts were prolonged briefly with 1,25-(OH)2D3 treatments (from 6 to 10 days). The dose of 1,25-(OH)2D3 necessary for this minimal graft prolongation was 500 ng/kg/day, given intraperitoneally. The overall conclusion from these experiments was that 1,25-(OH)2D3 only marginally prolongs graft survival.
In the accompanying article, we have shown prolongation of graft survival by feeding mice continuously with 50 ng/day/mouse of 1,25-(OH)2D3 (from 10 to 60 days) or 200 ng/day/mouse of 19-nor-1,25-dihydroxyvitamin D2 [19-nor-1,25-(OH)2D2] (from 10 to 56 days) or by feeding rats continuously with 500 ng/day of 1,25-(OH)2D3 (from 7 to 26 days) (3). Significantly, by 100 days after transplantation, 12% of the 1,25-(OH)2D3-treated and 22% of the 19-nor-1,25-dihydroxyvitamin D2-treated mice maintained actively beating heart muscle transplants. Cyclosporine (CsA), the standard against which evaluation of novel transplant drugs are often compared, when given at 25 mg/kg/day increased heart muscle allograft survival from 10 to 36 days (3).
For transplant patients, the two most serious side effects of CsA treatment are an increased susceptibility to infection and treatment-induced osteopenia (4-6). CsA given to mice at 25 mg/kg/day increased heart muscle transplant survival from 10 to 36 days. We have now studied the effects of these agents on bone mass and susceptibility of the host to infection. Candida albicans infections are one of the more common infections in immune-compromised individuals (4,7). Similarly, the members of the Herpes viridae family of viruses account for a large part of the morbidity and mortality of the immune compromised (4,8). Clinically important members of this family of viruses for transplant patients include herpes simplex virus (HSV), cytomegalovirus, Epstein-Barr virus, and human herpes virus-6 (4,8,9). Of considerable interest is that all human herpes viruses are frequently reactivated after immunosuppressive therapy for organ transplantation (8). To determine the susceptibility of host mice to infection, we used C albicans and HSV.
MATERIALS AND METHODS
Animals. Male C57BL/10 mice (Sprague Dawley, Indianapolis, IN) and female Balb/c mice (Sprague Dawley) were used for experiments when they were 7-8 weeks old.
Experimental diets/CsA treatments. Upon arrival from commercial vendors, mice were fed Purina Diet 5008 Formilab (Richmond, IN) containing vitamin D. For experiments, all of the mice were fed purified diets made in our laboratory as previously described (10,11). Mice were provided 4 g of the experimental diet per day (which was totally consumed). Food cups containing 8 g (for 2 days) or 12 g (for 3 days) and were replaced every 2-3 days for the duration of the experiment. Groups of 5-12 mice were fed the experimental diet (control treatment) or the experimental diet plus 50 ng 1,25-(OH)2D3/mouse/day. The diets were started 1 week before transplantation or infection. One group of mice on the control diet was also injected intraperitoneally with CsA (25 mg/kg/day). The 1,25-(OH)2D3 and the CsA treatments were continued for the duration of the experiments. The dose of CsA chosen was based on the reports of others (12). A dose of 2.5 mg/kg/day was too low to prolong graft survival in our experiments (3). The experimental diets contained 0.47% calcium and 0.3% phosphorus.
Although the experimental diet has no added vitamin D, the mice were exposed to fluorescent light, which provides for synthesis of vitamin D in the skin. In addition, vitamin D was provided in the Purina 5008 diet. Thus, the mice were not vitamin D deficient and must be considered as having adequate vitamin D.
Bone ash measurements. Mice were injected with 25 mg/kg/day CsA or provided 50 ng/day 1,25-(OH)2D3 exactly as was done to prolong graft survival (3). At day 50 after transplantation, all of the CsA-treated mice and 70% of the 1,25-(OH)2D3-treated mice had rejected their grafts, and the mice were killed for bone ash measurements (3). Femurs were collected from these mice treated with CsA or 1,25-(OH)2D3 for 6-8 weeks. The femurs were freed of adhering tissue and extracted with 100% ethanol for 24 hr. Excess ethanol was used to insure complete extraction. The femurs were then submerged in chloroform for an additional 24 hr to complete lipid extraction. Femurs were then dried in a 100°C oven for 12 or more hours and then weighed at room temperature. The femur dry bone weights reflect changes in the overall size of the mice. The femurs were then ashed in a 500°F muffle furnace for 24 hr. Ash weights were then determined at room temperature. Percent bone ash was obtained by dividing the bone ash value by the dry bone value and multiplying by 100. Percent bone ash values below 50% indicate that the mice have developed rickets.
C albicans infections. Groups of 5-6 C57BL/10 male mice were injected intravenously with 5×106_C albicans_ B311 (type A, kindly provided by Professor Edward Balish of the University of Wisconsin-Madison). Systemic candidiasis occurs in immune-compromised patients after dissemination of C albicans from the alimentary tract (5,7). Death due to systemic candidiasis is a result of overwhelming kidney infection, which leads to kidney failure (5,7,13).
HSV-1 infections. Female Balb/c mice (10 mice per group) were anaesthetized by inhalation of 3-5% halothane. The cornea was scratched three times vertically and three times horizontally with a 30-gauge needle, and a 5.0-µl drop of HSV-1 strain DRG4A1 virus suspension containing 106 plaque forming units was placed on the cornea (14). The drop was left for 30 sec, and the eye was closed. Any excess media was removed with a cotton swab. The DRG4A1 virus causes severe keratitis and, if not held in check by the immune system, will spread and cause viral encephalitis and death (14). Briefly, animals were anesthetized by inhalation of 3-5% halothane and scored for stromal keratis using a scale of 0-4. The severity of HSV-induced keratitis was scored as we have described previously: 0=normal; 1=cloudy iris, detail visible; 2=cloudy iris, detail obscured; 3=completely opaque; 4=corneal purferation (14).
Statistical significance. Statistical significance was determined using the Statview Student program for the Macintosh. A one-factor analysis of variance was used to compare dry bone weight, bone ash, and % ash. For Figures 1 and 2, statistical significance was determined using the two-sample test for binomial proportions (15). Values of _P_≤0.05 were considered significantly different.
Mortality of mice after systemic infection with C albicans. CsA-treated (●) 1,25-(OH)2D3-treated (▾), and control (◆) mice were infected intravenously with 5×106 C albicans. CsA significantly increased (_P_≤0.04) and 1,25-(OH)2D3 did not change the susceptibility of mice to systemic candidiasis. There were six mice in each group.
Mortality of mice after dissemination of HSV from the eye. Groups of 10-12 CsA-treated (●), 1,25-(OH)2D3-treated (▾), and control (▪) mice were infected in the eye with HSV. CsA caused the systemic spread and mortality of 100% of mice (_P_≤0.002), whereas 1,25-(OH)2D3 treatments and control mice showed similar mortality rates (36 and 30%, respectively). There were 10 mice in each group.
RESULTS
Bone ash measurements. CsA and 1,25-(OH)2D3 both induced changes in bone ash. Low-dose CsA caused a slight decrease in total bone ash values (Table 1), whereas the high-dose CsA, required for the prolongation of graft survival (3), caused a significant decrease (Table 1). The demineralization of bone was also reflected in the percent ash values. As expected, 1,25-(OH)2D3 treatments did not decrease total or percent bone ash but rather increased them. We conclude that CsA doses that result in transplant prolongation (25 mg/kg) induce bone loss, whereas 1,25-(OH)2D3 doses that result in transplant prolongation (50 ng/day) actually increase bone mass. It should be noted that all groups of animals had initially the same average body weights and hence ash values.
The effect of cyclosporin and 1,25-(OH)2D3 on bone ash_a_
Mortality after systemic C albicans infection. Groups of 6-10 C57BL/10 male mice were infected systematically with C albicans. This is the same mouse strain used as transplant recipients in other experiments (3). CsA treatment significantly increased the susceptibility of these mice to C albicans infection compared with the controls (_P_≤0.04) and the 1,25-(OH)2D3-treated (_P_≤0.007) animals (Fig. 1). Forty percent of the CsA-treated animals died after 9 days of infection, whereas none of either the controls or the 1,25-(OH)2D3-treated mice died. At 3 weeks after infection, only 20% of the CsA-treated mice were alive compared with 80% of the controls and 100% of the 1,25-(OH)2D3-treated mice. We conclude that 1,25-(OH)2D3 treatments do not increase the susceptibility of mice to C albicans infection. Mortality after ocular HSV infection. CsA treatment increased the susceptibility of Balb/c mice to HSV infection compared with the controls and the 1,25-(OH)2D3-treated animals (Fig. 2). One hundred percent of the CsA-treated mice were dead by 10 days after infection. Conversely, this viral infection disseminated and was lethal for only 30% of the controls and 36% of the 1,25-(OH)2D3-treated mice. Thus, 1,25-(OH)2D3 treatments do not increase the susceptibility of mice to HSV infection, whereas CsA clearly did.
HSV keratitis. To determine whether 1,25-(OH)2D3 treatment altered the severity of HSV-induced keratitis, we scored the severity of ocular disease. As shown in Figure 3, there was no difference in the disease scores between treated and control mice.
Mean disease scores for HSV-induced keratitis. Control (●) or 1,25-(OH)2D3-treated (▾) mice were infected with HSV-1 strain DRG4A1 and the severity of keratitis (corneal clouding) was scored on the days indicated. CsA-treated mice died before keratitis could be adequately scored and this group is not included. There were 10 mice in each group.
DISCUSSION
There is now little doubt that 1,25-(OH)2D3 has important effects on the immune system (16-18). The most impressive findings are that 1,25-(OH)2D3 completely blocks the development of at least two different experimental autoimmune diseases (16,17). Furthermore, the administration of 1,25-(OH)2D3 prolongs transplant graft survival much longer than the current drug of choice, CsA, in a mouse heart graft model (12). This in itself is surprising and intriguing. However, this might be expected if 1,25-(OH)2D3 is a general immune suppressant, as are the glucocorticoids and CsA. Indeed, 1,25-(OH)2D3 has been referred to as an immune suppressant (1,2). In our work with experimental autoimmune encephalomyelitis, it has become clear that 1,25-(OH)2D3 is selective in its actions on T lymphocytes in that it stimulates T helper 2 responses (18), while not affecting other immune cells. To pursue this hypothesis further, we examined the susceptibility of treated animals to infection by C albicans and HSV. The results showed, as expected, that CsA treatments markedly inhibit the mounting of an immune response to either C albicans or HSV, leading to the development of lethal infections. However, both controls and mice treated with 1,25-(OH)2D3 were able to resist these infections, illustrating that 1,25-(OH)2D3 is not a general immunosuppressant but a selective modulator of immunity. Further, these experiments suggest that the use of 1,25-(OH)2D3 to inhibit transplant rejection will not carry the same risks as CsA in increasing susceptibility to opportunistic infections.
HSV keratitis is an immunopathological manifestation of HSV corneal infection. Although there is some discussion concerning the underlying immunopathology resulting in HSV keratitis, it is generally considered to be due to the induction of a delayed-type hypersensitivity response (19). Delayed-type hypersensitivity responses are mediated by CD4+ T cells, and depletion of CD4+ cells from mice reduces or eliminates keratitis (19). Our observation that 1,25-(OH)2D3 had no effect on the severity of keratitis suggests that CD4+-mediated responses were not suppressed, lending support to the hypothesis that 1,25-(OH)2D3 is not acting as a general immunosuppressive agent.
Another problem facing transplant surgeons is that at least two major drugs used, i.e., CsA and glucocorticoids, to prevent transplant rejection, cause a loss of skeletal mass and endanger the patient to osteoporosis and fracture. We confirmed this finding in recipient mice treated with CsA. Not surprising is the fact that 1,25-(OH)2D3, while inhibiting transplant rejection, did not cause bone loss and instead increased total femur ash. Overall, the function of 1,25-(OH)2D3 is to improve calcium absorption, phosphorus absorption, and secondarily skeletal mineralization (20,21). Thus, a significant negative side effect of CsA and glucocorticoids could be averted with the use of 1,25-(OH)2D3 and its analogs as transplant antirejection agents.
A logical question might be: Why in previous studies did Lemire et al. (2) and Bouillon et al. (1) find minimal transplant protection from 1,25-(OH)2D3, whereas the studies from our laboratories show marked activity? Previous groups provided 1,25-(OH)2D3 by injection once a day or three times per week. Because 1,25-(OH)2D3 has a short lifetime, it is possible that biologically relevant concentrations of 1,25-(OH)2D3 were not maintained in vivo. By providing it in the diet, it is delivered in a more constant fashion, thereby being present continuously, accounting for superior activity. Another possibility is that the endogenous levels of 1,25-(OH)2D3, especially on a low-calcium diet, may be high and the amount contributed by the injection may have been relatively small.
This and the previous article (3) argue strongly in favor of pursuing 1,25-(OH)2D3 and its analogs for use as transplant antirejection drugs. Importantly, they should be provided in such a way as to be constantly available, as for example in slow-release form. Further, vitamin D analogs that are less apt to produce hypercalcemia should be given serious thought, because hypercalcemia is the major, if not sole, side effect of this form of therapy.
REFERENCES
1. Bouillon R, Okamura WH, Norman AW. Structure-function relationships in the vitamin D endocrine system. Endocr Rev 1995; 16: 200.
2. Lemire JM, Archer DC, Khulkarni A. Prolongation of the survival of murine cardiac allografts by the vitamin D3 analogue 1,25-dihydroxy-delta16-cholecalciferol. Transplantation 1992; 54: 762.
3. Hullett DA, Cantorna MT, Redaelli C, Humpal-Winter J, Sollinger HW, DeLuca HF. Prolongation of allograft survival by 1,25-dihydroxyvitamin D3. Transplantation (in press).
4. Aubia J, Masramon J, Serrano S, Lloveras J, Marinoso LL. Bone histology in renal transplant patients receiving cyclosporin. Lancet 1988; 1: 1048.
5. Kubak BM, Holt CD. Infectious complications of kidney transplantation and their management. In: Danovitch GM, ed. Handbook of kidney transplantation, 2nd ed. New York: Little, Brown and Company Inc., 1996.
6. Julian BA, Laskow DA, Dubovsky J, Dubovsky EV, Curtis JJ, Quarles LD. Rapid loss of vertebral mineral density after renal transplantation. N Engl J Med 1991; 325: 544.
7. Klein RS, Harris CA, Small CB, Moll B, Lesser M, Friedland GH. Oral candidiasis in high-risk patients as the initial manifestation of the acquired immunodeficiency syndrome. N Engl J Med 1984; 311: 354.
8. White DO, Fenner F, Herpesviruses. Medical virology, 3rd ed. Orlando: Academic Press, Inc., 1986.
9. Singh N, Carrigan DR. Human hypervirus-6 in transplantation: an emerging pathogen. Ann Int Med 1996; 124: 1065.
10. Yang S, Smith C, Prahl JM, Luo X, DeLuca HF. Vitamin D deficiency suppresses cell-mediated immunity in vivo. Arch Biochem Biophys 1993; 303: 98.
11. Yang S, Smith C, DeLuca HF. 1α,25-Dihydroxyvitamin D3 and 19-nor-1α,25-dihydroxyvitamin D2 suppress immunoglobulin production and thymic lymphocyte proliferation in vivo. Biochim Biophys Acta 1993; 1158: 279.
12. Babany G, Morris RE, Babany I, Kates RE. Evaluation of the in vivo dose-response relationship of immunosuppressive drugs using a mouse heart transplant model: application to cyclosporin. J Pharmacol Exp Ther 1987; 244: 259.
13. Cantorna MT, Woodward WD, Hayes CE, DeLuca HF. 1,25-Dihydroxyvitamin D3 is a positive regulator for the two antiencephalitogenic cytokines TGF-β1 and IL-4. J Immunol (in press).
14. Cantorna MT, Balish E. Acquired immunity to systemic candidiasis in immunodeficient mice. J Infect Dis 1991; 164: 936.
15. Brandt CR, Grau DR. Mixed infection with herpes simplex virus type 1 generates recombinants with increased ocular and neurovirulence. Invest Opthalmol Vis Sci 1990; 31: 2214.
16. Rosner, B, ed. Fundamentals of biostatistics, 2nd ed. Boston, MA: PWS Publishers, 1986: 584.
17. Cantorna MT, Hayes CE, DeLuca HF. 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis. Proc Natl Acad Sci USA 1996; 93: 7861.
18. DeLuca HF. Application of new vitamin D compounds to disease. Drug News Perspect 1992; 5: 87.
19. Doymaz MZ, Rouse BT. Immunopathology of Herpes simplex virus infections. In: Rouse BT, ed. HSV pathogenesis, immunobiology and control. Current topics in microbiology and immunology, Vol. 179. Berlin: Springer-Verlag, 1992: 121.
20. DeLuca HF. The vitamin D story: a collaborative effort of basic science and clinical medicine. FASEB J 1988; 2: 224.
21. DeLuca HF. The transformation of a vitamin into a hormone: the vitamin D story. In: The Harvey lectures, Series 75. New York: Academic Press, 1981: 333.
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