Immune dysfunction in hypophosphatemic vitamin D-resistant rickets: Immunoregulatory reaction of 1α(OH) vitamin D3

CLINICAL

IMMUNOLOGY

&ND

53, 24-31 (198%

IMMUNOPATHOLOGY

Immune Dysfunction in Hypophosphatemic Vitamin D-Resistant Rickets: Immunoregulatory Reaction of Ia Vitamin D, ISAO KITAIIMA,

IKURO MARUYAMA, HIROSHI MATSUBARA, AND AKIHIRO IGATA

MITSUHIRO

The Third Department of Internal Medicine, Faculty of Medicine. Kagoshima Us&i-cho, Kagoshima 890, Japan

Universio.

OSAME, 1208-I

We investigated immunologic function in six cases with hypophosphatemic vitamin D-resistant rickets (VDRR) before and after treatment with ln-hydroxycholecalciferol (la(OH) vitamin D,). All cases suffered frequent episodes of infection, which tended to be more severe in the older patients. OKT9-, OKTIO-, and OKMI-positive cells and adenosine deaminase (ADA) were significantly increased, whereas numbers and activity of natural killer (NK) cells were lower than normal before treatment. After administration of la(OH) vitamin D,, however, the susceptibility to infection apparently decreased, and NK cell number and activity increased in all patients. ADA was also significantly decreased and remained in the normal range after treatment. These results suggest that vitamin D plays a role in the impaired immunoregulatory functions of NK cells in VDRR. Furthermore, ADA may be one parameter reflecting this immunologic impairment. D 1989 Academic

Press. Inc.

INTRODUCTION

Hypophosphatemic vitamin D-resistant rickets (VDRR) is defined as “a specific disorder characterized by (a) hypophosphatemia associated with decreased renal tubular reabsorption of inorganic phosphate; (b) familial occurrence, the mode of inheritance being, most probably, sex-linked dominant; (c) the presence of rickets or osteomalacia which does not respond to the usual vitamin D treatment; and the absence of other related abnormalities” (1). It is presumed that the disorder is the result of abnormal metabolism of vitamin D and that large doses of la-hydroxycholecalciferol (la(OH) vitamin D,) can improve such symptoms as bone lesions and hypophosphatemia (1). While much attention has been focused on the symptoms and pathogenesis of VDRR. complications in untreated adult cases have yet to be fully described (2). We have examined several untreated adult cases of VDRR and observed frequent episodes of infection. Vitamin D, originally discovered to function as an antirachitic agent (3), has been studied regarding its roles in controlling calcium metabolism. Vitamin D is known to stimulate differentiation of HL-60 cells, a human leukemic cell line, into macrophages (4); hence, there has been a recent focusing of attention on the immunoregulating effects of vitamin D (5). These effects are exemplified by the increased susceptibility to infections of patients with abnormalities in vitamin D metabolism of renal tubules with associated renal insufficiency (6). In certain cases, vitamin D deficiency is also known to be associated with frequent episodes of infection (7). 24 0090-1229/89 $1.50 Copyright All rights

0 1989 by Academic Presr, Inc. of reproduction in any form reserved.

IMMUNE

DYSFUNCTION

IN VDRR

AND

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TO VITAMIN

25

D

In the present study, we investigated the immunological functions of six untreated patients with VDRR and evaluated the immunological effects of administration of la(OH) vitamin D,. MATERIALS

AND METHODS

Patients and Control Donors The study population consisted of six VDRR cases (3 males and 3 females) in three families. The diagnosis of VDRR was established by both clinical and laboratory findings. These findings were short physical stature, hypophosphatemia, characteristic abnormalities in bone roentgenogram, and hyperosteoidosis in bone biopsy. The profiles of all six cases are summarized in Table 1. All cases were studied before and after a 4-month administration of la(OH) vitamin D,. The study began after cessation of lol(OH) vitamin D, administration for more than 3 months for the purpose of washing out the medicine. Fifty healthy individuals (26 males and 24 females, age range: 10-68 years, mean + SD: 36 ? 18 years old) served as controls. Laboratory Tests (a) Immunoglobulin G, A, and M (IgG, IgA, and IgM) measurement by single radial immunodiffusion. (b) Total complement activity (CHSO) determination by the method of Mayer (8). C3 and C4 proteins were measured by single radial immunodiffusion using antisera against these components obtained from the Berringwerke Institute (Marburg, West Germany). (c) Lymphocyte-surface phenotype identification using murine monoclonal antibodies against OKT3 (all peripheral blood T cells), OKT4 (inducer/helper T cells), OKT8 (suppressor/cytotoxic T cells), OKT9 (transferrin receptor and activated T cells), OKTlO (precursor cells and activated T cells), OKIal (Ia antiVDRR

TABLE PATIENT

1 PROFILES Family number

Age Sex

Height (cm) Serum Ca (mgidl) P (mgidl) PPD test* Episodes of infection

Doses of Ia(O (w&W * Purified

A I

A 2

A 3

B 1

B 2

C 1

9 years old Male 102

17 years old Female 110

24 years old Male 122

28 years old Male 126

55 years old Female 125

56 years old Female 118

90 I .4

8.8 1.5

8.7 1.5

8.9 1.4

9.2 1.6

9.0 1.5

(+I

(+I

Common cold

(+)

Common cold. Cystitis

Common cold. Cystitis

2.0

4.5

5.5

protein derivate

of tuberculin

test.

(21 Common cold, Bronchitis, Cystitis

6.0

(-1 Common cold, Pneumonia, Cystitis

2.5

C-b Common cold, Pneumonia, Filariasis

3.0

36

KITAJIMA

ET

AI

gens), and OKMl (null cells, monocytesimacrophages. and granulocytes). These antibodies were supplied by Ortho Co. Ltd. Leu7 and Leul I (NK cell) were supplied by Becton Dickinson. Briefly, 5 ~1 of the appropriate monoclonal antibody (50 mgiml) was added to 200 pl of the cell suspensions at 4°C for 30 min. The cells were then washed three times with phosphate-buffered saline (PBS), pH 7.4. at 4°C after which 100 ~1 of a 1: 100 dilution of fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG was added to the cell pellet at 4°C for another 30 min. The cells were then washed with PBS, pH 7.4, at 4°C and the percentage of positive cells was determined by flow cytometry (Spectrum III. Ortho Co. Ltd.) (g-12). (d) NK cell activity was measured by a standard “Cr release assay on KS62 target cells using a modified version of the method of Rimm and co-workers ( 13). “Cr-labeled target cells (T) were coincubated with the effector cells (E). peripheral blood mononuclear cells, in plastic tubes for 4 hr (E:T = 40: 1). Radioactivity measurement was performed using a Beckman 4000 y scintillation counter and the percentage of “‘Cr release was calculated (percentage lysis = [sample count spontaneous count/maximum count - spontaneous count] X 100). (e) The ADA activity assay was performed using a modified version of the method by Giusti (14). This method is based on the determination of ammonia released from an enzyme reaction (adenosine + HzO, -+ inosine + NH,), released ammonia by the indophenol reaction. Incubation was carried out at 37°C for 30 min with a 50-p,l sample and 200 pl of buffered substrate (6 mM adenosine, 0.05 M phosphate buffer, pH 6.5). The reaction was terminated by addition of I .S ml of a phenol solution (106 mM phenol, 0.17 mM sodium nitroprussid). Two milliliters of alkaline hypochlorite solution (0. I7 mM NaOCI, 125 mM NaOH) was then added and incubated for 20 min at 37”C, after which the developed blue color of the indophenol complex was measured in a spectrophotometer at 630 nm. In the same manner, a blank sample (without adenosine), a blank reagent (distilled water without sample and adenosine), and an ammonia standard solution (NH,. 20 pg/ml) were assayed. ADA activity was expressed in international units (IU) per liter. All tests were repeated 3 months after the normalization of serum phosphate in response to the administration of 3-6 &day of Ia vitamin D,, the doses of which are shown in Table I. These cases were followed up for another year. All tests were also performed on the 50 normal controls. RESULTS All six VDRR patients exhibited frequent episodes of infection. These included common cold, cystitis, bronchitis, pneumonia, and filariasis (Table 1). This tendency appeared to be more severe in the older patients. The patients of one decade had episodes of frequent infections such as bronchitis and tonsillitis. The two oldest patients in the study had histories of repeated severe viral pneumonia requiring hospitalization. The purified protein derivate of the tuberculin test (PPD) of both patients proved negative (Table I). Before la(OH) vitamin D, administration, the lymphocyte and the monocyte

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levels of all patients were within normal limits (Table 2). IgA, IgM, and the complement system were also normal. IgG was slightly lower than normal. On the other hand, OKT9-, OKTlO-, and OKMl-positive cells were significantly increased (P < O.OOS), while the NK cell numbers representing Leul l-positive cells and NK cell activity were markedly decreased (P < 0.005) when compared to normal controls. ADA was significantly increased in all six cases (mean + SD: 29.3 ? 7.5, normal range; 13.8 + 4.4, P < 0.005). The ADA values in the two older patients were higher than those of the other patients (41, 30 Ill/liter). The immunological changes after administration of lol(OH) vitamin D, are shown in Table 2 and Figs. l-3. Administration of lo(OH) vitamin D, generally showed favorable effects, such as improvement of bone lesions. Furthermore, negative PPD test in the two oldest patients (B-2 and C-l in Table If became

SUMMARYOF

IMMUNOLOGICAL

TABLE 2 STUDIESBEFOREANDAFTERTREATMENTWITH D, IN VDRR ANDNORMALCONTROLS VDRR Before

~_ ~~~_ _~ Serum Ca (mg/dl ) P (mg/dl) lu,25(OH), Vitamin D, (pg/mlt WBC Lymphocyte Monocyte Complement CH50 (CHSO/ml) c3 (mg/dl) c4 (mg/dl) lmmunoglobulin 1s (mg’dl) kA (mg/dl) W (mg/dl) T cell subsets OKT3 (%) OKT4 WC) OKT8 VCF) OKT4/OKT8 (%) OKT9 (%) OKTIO (%‘c) OKIaI (%) OKMI (%) Leu7 (%i) Leul I (%I Activity of NK cells (%I ADA (IU/liter) * P ‘< 0.005.

treatment

8.93 2 0.16 1.48 t- 0.07’

-_.

patients

(mean

la(OH)

2 SD) After

treatment

9.37 -e 0.84 2.55 t- 0.35

VITAMIN

Controls (mean + SD)

9.52 t 0.11 3.42 t 0.82

20.5 2 8.4*

71.5 f IO.1

48.6 t

7480 2 2049 3825 +- 1556 214 t 77

6400 t 1098 3716 t 1158 246 ~tl 78

6366 t 1734 3565 t 847 256 t 175

38.5 2 6.7 76.5 + II.0 29.0 -t 9.4

40.6 1- 7.3 71.3 t 9.7 29.8 t- 8.0

34.8 c 5.2 81.3 t 31.2 32.8 2 13.2

1048 + 149 245 2 30 I05 2 28

1054 t 163 242 t 36 100 t 26

1340 t 540 277 t 185 II9 t 51

75.5 38.5 28.9 1.38 1.08 21.9 23.4 21.8 18.2 9.1

74.3 37.3 28.9 1.34 1.08 23.3 22.8 30.5 1X.6 13.8

69.5 38.1 28.8 1.35 0.26 10.3 17.6 11.6 18.4 16.1

+ 6.2 11.7 k 4.2 + 0.38 2 0.55* 2 5.6* 2 6.4 + 8.6* + 9.6 i 3.9*

2

19.3 + l1.8* 29.3 f 7.5*

k t t + t

t t t t

rfr

7.7 II.0 4.5 0.39 0.53* 6.4* 6.5 6.2* 9.2 5.4

26.0 t 14.1 18.8 t 7.3

17.7

+ 6.1 2 5.8 t 6.5 t 0.45 t 0.24 t 5.7 t 6.7 t 3.7 f 5.9 ? 6.7

32.5 t 14.8 13.8 t 4.4

28

KITAJIMA

E’I Al

positive after la(OH) vitamin D, therapy. Although the numbers of OKT9- and OKTI O-positive cells remained unchanged (Fig. I ), OKM 1. Leu 1I -positive cells and NK cell activity increased in all cases (Fig. 2). ADA, which was elevated in all cases before treatment, significantly decreased (0.005 < P c 0.011, and in three cases, even dropped to within normal levels after administration of Ia vitamin D, (Fig. 3). There was no significant change in immunoglobulins or the complement system after the treatment. During the minimum l-year period in which all cases were followed up with administration of Ia vitamin D,, no cases exhibited a common cold or other infections. DISCUSSION

All six VDRR patients investigated had previous histories of recurrent infection. The susceptibility to infection was more severe in the older patients. This observation is further confirmed by the negative PPD test of the two oldest patients. These clinical observations enabled us to investigate immune function in VDRR patients in a manner previously undescribed. The most remarkable abnormality observed in this study was the high value of ADA, reflecting lymphocyte metabolism (15). Increased ADA has been described OKT

g

OKT

10

% 40

30

20

...... ...... .-.- .*:.-.-.-. ...... ::::.* .*::::, ‘::::: ................... ................ .......... . . :.x... ......... ......... .................... :.:.: :.....:.......:.:.:.:.: .: ..... “:.‘.*.-.* .......... .A% ...... ...... ...... ..... . .;;.A<.%~.%~.y :y :~+x(.~{ ............. ............ ............ . ...................... ............ .:. ....................... . .......... .......... . . .... . ............. .......... .......... .............. ......... .. ... . . .............. ............. ........ ....... . . ... . .............. ............. ....... ...... .- ... ... ............. “:.*.*.-.* .... .... .............. ............. ...... ...... ...... .............. .............

10 ~

0

0

BEFORE

AFTER

l----l-

FIG. 1. OKT9- and OKTlO-positive cells. Closed circles indicate patients with VDRR (n = 6): shaded areas indicate control values (mean k SD, n = 50). “Before” means before treatment with lu(OH) vitamin D, and “after” means 2 months after normalization of serum phosphate in response to administration of la(OH) vitamin D,.

IMMUNE

DYSFUNCTION

OKMI

IN VDRR

AND

Leu7

0.06
EFOR

Leull

O.l
25

RESPONSE

0.1
TO VITAMIN NK

Cell

D

29

Activity

0.05
25

AFTEI i

FIG. 2. OKMI, Led, Leull, and NK cell activity. Closed circles (T cell subsets) and closed triangles (NK cell activity) indicate patients with VDRR (n = 6); shaded areas indicate control values (mean -t SD, n = 50). “Before” means before treatment with la(OH) vitamin D, and “after” means 2 months after normalization of serum phosphate in response to administration of la(OH) vitamin D,.

in acute lymphoblastic leukemia (16), adult T cell leukemia, and infectious mononucleosis and tuberculosis (17). Here we report that ADA increases in VDRR. Since ADA is known to increase in acquired immune deficiency syndrome (AIDS), it is possible that the high level of ADA in VDRR is due to impaired immune function. Provvedini et al. (18) reported that la(OH) vitamin D, has effects on B lymphocytes and suppresses production of immunoglobulins. However, immunoglobulins, including IgG, were not significantly affected by the administration of lor(OH) vitamin D, in our cases. This discrepancy might depend on the difference cells, however, between in vivo and in vitro studies. OKT9- and OKTIO-positive were observed to be increased before treatment, suggesting the occurrence of T cell activation in VDRR. On the other hand, both Leul l-positive cells, reflecting NK cell number, and NK cell activity were found to be decreased. As VDRR is defined as a disorder of vitamin D metabolism, we investigated the relationship between these immunological disorders and vitamin D metabolism. The administration of bx(OH) vitamin D, resulted in a decreased susceptibility to infections with normalization of NK cell activity and NK cell number. The increased ADA levels observed in all cases also responded to treatment with

30

KITAJIMA

t-.1

Al

ADA 0.005
r--

IU/I

7

I 1 I

0

-I-BEFORE

I I I I

AFTER

FIG. 3. Adenosine deaminase (ADA). Closed circles indicate patients with VDRR (n = 6); shaded areas indicate control values (mean + SD. n = SO). “Before” means before treatment of Ia vitamin D, and “after” means 2 months after normalization of serum phosphate in response to administration of Ia vitamin D,.

la(OH) vitamin D, and actually normalized in three patients. From these results, vitamin D appears to act via the monocyte/macrophage system and/or NK cells in effecting its immunoregulatory functions in VDRR. Furthermore, the fact that increased ADA was significantly decreased upon administration of lo(OH) vitamin D, suggests that ADA may be an additional abnormal laboratory finding in VDRR. Concerning the relationship between immune function and bone metabolism, Fujita et al. (19) reported that patients with advanced osteoporosis had a predominance of negative PPD tests and increased OKT4/OKT8 ratios, though both factors normalized after treatment with Ia vitamin D,. These findings parallel our results in VDRR; hence, we presume that vitamin D is capable of regulating immune functions. This hypothesis is further confirmed by the reports of various investigators regarding in vitro studies. Nakao er al. (20) reported that HL-60 cells differentiated to OKMl-positive cells in proportion to the concentration of la,25 dihydroxycholecalciferol (la,25(OH), vitamin D,). Matsui ef al. (21) also demonstrated that lo1,25(OH), vitamin D, facilitated the differentiation of HL-60 cells to monocytes and activated macrophages. Recently, Minghetti and Norman (22) reviewed the multiple functions of lq25(OH), vitamin D3 at the molecular level. They described that the actions of la,25(OH), vitamin D, appear to be mediated through a hormone receptor complex and that vitamin D, functions as a steroid hormone in the differentiation and activity of lymphocytes and monocyte/macrophages.

IMMUNE

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D

31

In this study, we demonstrated immune dysfunction in VDRR. This immunoLogic abnormality responded to the administration of la(0I-I) vitamin D3. Thus, we conclude that vitamin D acts not only on calcium metabolism but also on immune functions. ACKNOWLEDGMENT We are grateful for the help of Dr. Raymond L. Resales in the preparation of this paper

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Burnet. C. H.. Dent, C. E.. Harper, C.. and Warland, B. J.. Amer. J. Med. 36, 222, 1964. Yoshikawa. S., Shiba. M.. and Suzaki, J. Bone. ft. Srtrg. 50A, 743. 1968. McCollum, E. V., Simmonds, N., Shipley, P. G.. and Prak, E. A., J. Biol. Chem. 51, 41, 1922. Tanaka. H.. Abe. E., Miyaura, C.. Kuribayashi. T.. Konno. K., Nishii, Y.. and Suds, T.. Biothem. .I. 204, 713, 1982. Dowling. G. S., and Prosser Thomas. E. W., Larwer 1, 919, 1946. Lawrence, H. S.. Ann. Intern. Med. 2, 166, 1965. Lorento. F., Fontan. G., Jara, P.. Casa. C.. Garcia-Rodriguez, M. C.. and Ojeda. J. A.. Acta Paediarr. Stand. 65, 228. 1970. Mayer, M. M.. “Experimental Immunochemistry” (E. A. Kabat and M. M. Mayer. Eds.). p. 133. Thomas. Springfield, IL. 1961. Thomas, Y., Sosman, J.. Irigoyen. 0.. Friedman. S. M.. Kung. P. C.. and Goldstein. G.. J. fmmunol. 125, 2402, 1980. Friedman. S. M., Hunter. S. B.. Irigoyen. 0.. Kung. P. C., Goldstein. G.. and Chess, L.. J. Immunol. 126, 1702, 1981. Thomas. T., Sosman, J.. Rogozinski. L.. lrigoglu. 0.. Kung. P. C.. and Goldstein. G.. J. Immrtnol. 126, 1948. 1981. Barnaba, V., Zaccari. C.. Levrero. M.. Ruocco. G.. and Balsano. F.. C/in. fmmrtnol. Immunopathol. 26, 83, 1983. Rimm. I. J., Schlossman, S. F.. and Reinherz. E. L.. Cell. Immrrnol. 87, 327. 1984. Giusti, G.. Methods Enzym. Anal. 2, 1092. 1974. Hovi. T. J. F.. Smyth. A. C., Allison. A. C., and Williams. S. C.. C/in. Erp. Immunol. 23, 395. 1976. Smyth. J. F.. Poplack. D. G.. Holiman. B. J., Leventhal. B. G.. and Yarbro. G.. J. C/in. fnr*e,sr. 62, 710, 1978. Muller-Beissenhirtz. W.. and Keller. H., Drsch. Med. Woc,henschr. 91, 159, 1966. Provvedini. D. M., Tsoukas. C. D.. Deftos. L. J.. and Manolagas. S. C., J. Immunoi. 136, 2734. 1986. Fujita. T., Matsui, T.. Nakao, Y.. and Watanabe. S., Miner. E/ec~tro/yre Metah. 10, 375, 1984. Nakao. Y.. Matsui. T., Koizumi. T.. Nakagawa. T., Katakami, Y.. and Fujita, T.. J. Nufr. Sci. Vitaminol. 31, 49, 1985. Matsui. T.. Takahara. R.. Mihara, K., Nakagawa. T.. Koizumi. T.. Nakao. Y.. Sugiyama, T.. and Fujita. T., Cancer Res. 45, 4366. 1985. Minghetti. P. P.. and Norman, A. W.. FASEB J. 2, 3043. 1988.

Received November 29. 1988: accepted with revision April 17. 1989