malignant osteoporosis and defective immunoregulation

JOURNAL OF BONE AND MINERAL RESEARCH Volume 3, Number 5, 1988 Mary Ann Liebert, Inc., Publishers

Malignant Osteoporosis and Defective Immunoregulation

KAREN R. RUBIN,' MARK BALLOW, ROLAND BARON, ROBERT M. GREENSTEIN, LAWRENCE G. RAISZ, and DAVID W. ROWE

ABSTRACT

The linkage between immune cells and the osteoclast has become partially understood in the laboratory, but the full spectrum of clinical disorders of this relationship remain to be elucidated. We report a 29-month-old girl with recurrent infections and multiple fractures. Immune evaluation showed normal quantitative serum immunoglobulins but absent antibodies to the respiratory viruses and tetanus toxoid and decreased in vitro polyclonal-induced immunoglobulin production. Further analysis in vitro with separated lymphocyte populations showed normal B cell function but markedly increased suppressor T cell activity. The bone evaluation showed diffuse osteopenia on x-ray. Serum calcium, phosphorus, PTH, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D were normal for age. Urinary calcium excretion (24 h) was, however, two times normal. An iliac crest biopsy confirmed the presence of extreme osteopenia with normal mineralization and numerous small atypical osteoclasts resorbing the bone. No circulating plasma resorptive activity was dem- onstrated. Calcitonin therapy markedly diminished the patient's hypercalciuria. We speculate that this patient's increased bone resorption, decreased bone formation, and suppressor activity may be linked by a common pathway involving the abnormal function of immune cells. Since no similar constellation of findings has been previously reported, this case may represent a new congenital disorder: severe osteopenia associated with increased osteoclast activity in association with a defect in T cell immunoregulation.

INTRODUCTION

HE DEMONSTRATION that a transplant of histocompati- T ble marrow cells can correct the bone-resorbing defect found in osteopetrosis provided a firm link between the marrow stem cells and the osteoclast."' Subsequently other observations have underscored the relationship between immunoregulatory cells and the osteoclast. A bone- resorbing factor obtained from mixed cultures of monocytes and lymphocytes (osteoclast activating factor) has been implicated in the pathogenesis of hypercalcemia and bone resorption in patients with multiple myeloma.'*' Re- cently, a comparable bone-resorbing factor was shown to be a product of the interaction between T lymphocytes and macrophages.I3) 1,25-dihydroxyvitamin D, a stimulator of

bone resorption by osteoclasts in vivo, can induce the differentiation of a human monocyte leukemia cell line (HL60) to an osteoclasticlike multinucleated Recent data suggest that the production of T cell lymphokines, which can modulate the differentiation of the monocyte to an osteoclast, may also be regulated by 1,25-dihydroxyvitamin D. ( 5 ) Interleukin-1 (IL-1), a monocyte product produced during inflammation, has been shown to stimulate bone resorption in vitro. Finally, monocyte-derived tumor necrosis factor (TNF-a) and lymphocyte-derived tumor necrosis factor (TNF-b) have been found to stimulate bone resorption and inhibit bone formation in vitro."' Thus, multiple pathways link the immune system to the process of bone resorption.

If the cells of the immune system play a significant role

'Department of Pediatrics and Medicine, University of Connecticut, Farmington, C T 06032. 2Department of Medicine, Yale University, New Haven, C T 06511.

509

510 RUBIN ET AL.

in the activity of the osteoclast, then clinical disorders of this relationship can be anticipated. Certain forms of osteopetrosis may reflect an abnormality in the Jiiferentia- tion of the monocyte or osteoclast.(*) In this paper, we report a case of osteopenia secondary to hyperplasia of the osteoclast in association with a defect in T cell iwnunoreg- ulation. Since a similar constellation of findings has not previously been reported, this case may represent a newly recognized congenital disorder.

CASE HISTORY

K.D. presented to the endocrine service at the University of Connecticut at 29 months of age for evaluation of multiple long bone fractures. She was born to a 28-year-old gravida 10 para 6, A3, L6 mother by cesarean section due to maternal fever at 34 weeks of gestation. She required supplemental O2 for 98 h for mild respiratory distress syndrome. Because several family members had phenylketo- nuria, this disorder was diagnosed in this patient in the first 2 weeks of life for which she was begun on appropriate dietary management. During the first several years of life she had multiple episodes of otitis media, impetigo, conjunctivitis, and pneumonia. Outpatient follow-up at the University of Connecticut Genetics Clinic documented significant neurodevelopmental delay accompanied by sei- zures and failure to thrive. Her initial fracture of the humerus occurred at 1 year of age while being manipulated for an upper gastrointestinal series performed to investi- gate her episodes of recurrent vomiting. Over the next year she sustained 15 fractures of the extremities, including one of the humerus during a venipuncture.

Physical examination revealed a deaf, nonverbal, cachectic, white female encased in four fiberglass extremity casts. She had severe growth retardation, measuring 75.5 cm (less than third percentile) and weighing 8.5 kg (less than third percentile). Her short stature was proportional. She had sparse blond hair, fine facial features, and bluish sclera. There were bilateral hemorrhagic exudates present in the external ear canal. There were no dysmorphic facial features, no unusual scarring or joint laxity, and no rachitic rosary. There was no family history for fractures or other connective tissue abnormalities.

General laboratory studies revealed a hematocrit of 40%, white blood cell count of 9500 with a normal differential, and a platelet count of 302,000. The sodium was 136 mEq/liter, potassium 3.6 mEq/liter, chloride 101 mEq/liter, carbon dioxide 23 mEq/liter; blood urea nitro- gen 10 mg/dl, and creatinine 0.8 mg/dl. The total protein was 6.1 g/dl (normal 6.1-7.7), and albumin was 4 g/dl (normal 3.8-4.9). Thyroid function tests were consistent with a heritable thyroxine binding globulin (TBG) deficiency and normal thyroid status (thyroid stimulating hormone, TSH, 2.4 mU/ml; T,, 4.5 pg/dl, and T, resin up-

take ratio 1.30). The morning serum cortisol was 20 pg/dl, and the 24 h urinary-free cortisol was 36 pg/day (normal < 100 &day). Total serum calcium was 4.3 mEq/liter (normal 4.8-5.8 mEq/liter), but the free calcium determined by ultrafiltration was 3.3 mEq/liter (normal 2.2- 3.2) and the ionic calcium determined by a calcium ion analyzer was 2.6 mEq/liter (normal 2.2-2.6). A 24 h urine for calcium excretion was twice the expected value for her size: 5.9 mEq/day or 0.7 mEq/kg per day (normal 0.4 mEq/kg per day). The serum alkaline phosphatase was 227 unitdm1 (45-300 U/ml). The C-terminal immunoreactive PTH was 59 p1 Eq/ml, and a repeat level was 78 pl Eq/ml (Nichols, normal 40-100 pl Eq/ml); however, the urinary cyclic AMP was greatly elevated: 15-30 mM/g creatinine (normal 2.2-5.9). Serum concentrations of the vitamin D metabolites were normal: 25-hydroxyvitamin D, 15 ng/ml (normal 9-43 ng/ml); 1,25-dihydroxyvitamin D, 81 pg/ml (normal 23-1 10 pg/ml). X-rays of the long bones demon- strated multiple fractures in varying stages of repair and diffuse and severe osteopenia but no changes typical of rickets or hyperparathyroidism (see Fig. 1).

The routine immunologic evaluation, performed to ex- plain the recurrent episodes of pyogenic infections, showed age-appropriate serum concentrations of immunoglobulins: IgG, 700 mg/dl; IgM, 100 mg/dl; and IgA, 5 5 mg/dl. IgC subclasses were IgG,, 355 mg/dl; IgG2, 40 mg/dl; IgC,, 92 mg/dl; and I&, 12 mg/dl, which are all within normal range for her age. Isohemagglutinins were positive to blood group A (1-16) and blood group B (1-32). The tetanus antibody titer was low despite adequate immuniza- tion (19% of normal adult controls). An antibody screen for the respiratory viruses showed the absence of specific complement-fixing antibodies to influenza, adenovirus, mycoplasrna, and respiratory syncytial virus. The total lymphocyte count ranged between 6700 and 7800 mm-3.

During the terminal phase of the patient’s illness, the major focus in her management was in establishing adequate protein and caloric nutrition in the face of recurring episodes of vomiting and in immune reconstitution with intravenous immune serum globulin. This required long- term parenteral hyperalimentation, which was complicated by bacteremia, fungemia, and other opportunistic infections. The patient’s recurrent hemorrhagic otitis and mas- toiditis were ultimately resolved by a bilateral mastoidect- omy. The surgery revealed such extensive erosion of the bone structures of the middle ear that the left carotid artery was exposed.

At 40 months of age the patient died. An autopsy did not reveal an immediate cause of death. However, the autopsy findings did confirm the presence of changes consistent with this child’s malignant osteoporosis. The extremities showed the absence of normal bone rigidity. Mi- croscopic examination of the kidneys showed the presence of numerous calcific deposits within the convoluted tu- bules, a finding consistent with the presence of hypercalciuria, which may have been associated with nephrocalcino- sis. Four parathyroid glands were identified and examined and showed no evidence of hyperplasia or tumor.

OSTEOPOROSIS AND lMMU NOREGULATION 511

8

FIG. 1. General view of the iliac crest biopsy showing the extremely thin cortices and low trabecular bone volume.

METHODS Collagen analysis

Dermal fibroblasts were established to evaluate the possibility of osteogenesis imperfecta. The cells were grown in Dulbecco's minimum essential medium containing 10% horse serum, 100 U/ml penicillin, and 100 &liter strepto- mycin. Upon reaching visual confluency, the cells were ra- diolabeled in the presence of 25 pg/ml of ascorbic acid with 10 pCi/ml of [5-'H]proline for 6 or 24 h followed by extraction of the cell and media proteins as previously described.(8) Total collagen was determined by the collagenase digestion procedure of Peterkofsky and Deigel- mann,(9) and the relative production of type I and type 111 collagen was obtained from interrupted sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis. ( l o )

In vitro bone resorption assay

Analysis of the serum for a circulating bone-resorbing factor employed the method described by Trummel et al.(") In this bioassay, limb bones from fetal rats previ-

ously exposed in utero to 4sCa were cultured in modified BGJ media containing either 10% patient or control sera. The labeled bones were also incubated with the supernatants of phytohemagglutinin-stimulated lymphocytes from the patient or control individuals. The amount of resorption of T a from the long bones was measured by the release of the isotope into the media during a 5 day culture period. The amount of radioactivity, expressed as a percentage of the total radioactivity initially present in the tissue, is used as a measure of the bone-resorptive activity of a sample.

All calcitrophic hormone measurements were performed by the Nichols Institute Reference Laboratories.

Bone biopsy

A transcortical iliac crest biopsy was performed under local anesthesia 2 cm inferior and 2 cm posterior to the an- terosuperior iliac spine. The biopsies were fixed in 40% ethanol at 4"C, dehydrated through graded alcohols, and embedded without decalcification in methylmethacry- late.(L2) Sections 4 p thick were obtained with a Jung K

RUBIN ET AL. 512

sliding microtome and stained with toluidine blue (pH 3) and Von Kossa or Goldner trichrome. Biopsy material from the same anatomic area was obtained from normal children in the same age range (two acute accident victims). Histomorphometric measurements were made using a planimeter (MOP 3, Zeiss, Germany) as previously described. (I3)

Immunologic studies

Serum immunoglobulin and IgG subclasses were quantitated by the clinical laboratories using nephrolometry and radial immunodiffusion, respectively. The proliferative response of peripheral blood mononuclear cells (PBM) to mitogens and specific antigens were performed as previously de~cribed."~) T cell subsets in the peripheral blood were analyzed by indirect immunofluorescence using monoclonal antibodies from Ortho Diagnostic Systems (Raritan, NJ).

Pokeweed mitogen (PWM)-induced B cell immunoglobulin (Ig) synthesis was ascertained by methods as previously de~c r ibed .~ '~ ) Ig isotypes in the culture supernatants were quantitated by an enzyme-linked immunoabsorbent assay (ELISA) according to the methods of Wasserman et a1.(16) Using a standard curve derived from human serum containing known amounts of IgG, IgM, and IgA (Calbio- chem-Behring, LaJolla, CA), a linear regression analysis was performed on the linear portion of the standard curve. The quantities of Ig produced during the 9 day culture period were calculated by polnomial regression and expressed as nanograms per milliliter of supernatant fluid.

To evaluate abnormalities in immunoregulation, specif- ically suppressor activity, equal numbers (1 x lo5) of mononuclear cells from the patient and a normal individ- ual were cocultured in microtiter plates as previously described."'' The degree of suppressor activity was determined as follows:

observed Ig synthesis in coculture Vo change = - 1 x l o o expected Ig synthesis from

patient and control PBM cultured alone

A negative value denotes increased suppressor activity. Co- culture of control pairs ( N = 25) never gave a percentage change above -25. The assessment of suppressor activity by macrophages was determined by the addition of indomethacin (1 g/ml) to cell cultures.'18)

RESULTS

The patient's initial blood chemistries and x-rays ruled out the various rachitic and/or osteomalacic disorders of in- fancy and childhood as well as primary or secondary hyperparathyroidism. Similarly, the initial studies eliminated hypercortisolism and hyperthyroidism as a cause of the patient's fractures. We therefore considered osteogenesis im-

perfects. Routine collagen typing performed on the patient's cultured fibroblasts showed normal total collagen production and a normal ratio of type 111 to type I collagen.

An initial biopsy was performed at 29 months of age. Tet- racycline labeling was not done. The cortex appeared thin and did not show the periosteal bone formation typical in a child this age (Fig. l), and trabecular bone volume was markedly reduced. Furthermore, a mineralization defect of the bone matrix was not evident. The most important finding was the presence of numerous small osteoclastic cells on the endosteal surface of the cortex and the trabecular surface (Fig. 2). The postmortem biopsy performed at 40 months of age showed an extremely low trabecular bone volume and confirmed the previous morphological observa- tion of numerous small osteoclasts with one to two nuclei.

On the basis of the initial bone biopsy findings of a hyperresorptive state and the patient's well-documented hypercalciuria, we instituted a trial of calcitonin for both diagnostic and potentially therapeutic purposes. As can be seen in Figure 3, the calcitonin diminished the patient's hypercalciuria but did not result in obvious clinical improve- ment. The drug was discontinued at the lowest effective dose because it aggravated her persistent vomiting.

Because the initial immune studies suggested a deficiency in the production of functional antibodies to common viral pathogens, in vitro studies were performed to further deline- ate the nature of the immune defect. Analysis of T cell subsets using monoclonal antibodies showed 90% T3-positive cells, 55% T4-positive cells, and 36% T8-positive cells, the latter T8 subpopulation being very high. The patient dem- onstrated only 3% B lymphocytes, which stained for only IgG and IgM. The patient's lymphocytes responded nor- mally to the T cell mitogens phytohemaglutinin (PHA) and concanavalin A. Normal lymphocyte proliferative responses to several specific antigens, including candida, tetanus, and diphtheria, were present.

PWM-induced polyclonal B cell activation of whole peripheral blood mononuclear cells showed severe impairment in the synthesis of all three immunoglobulin classes (Table 1). On several occasions, coculture of the patient's mononuclear cells with control mononuclear cells from different individuals showed the presence of increased suppressor activity. This suppressor activity was not abrogated by the re- moval of monocytes or by the addition of indomethacin to cell cultures. Lymphocyte separation experiments were performed to study the abnormalities in immunoregulation further. Coculture of the patient's B cells with control T cells resulted in the ability of the patient's B cells to synthesize immunoglobulins (Table 2). In contrast, coculture of control B cells with the patient's T cells showed diminished synthesis of all three immunoglobulin classes. The latter results, together with the monocyte depletion and indomethacin studies, suggested that this increased suppressor activity re- sided in the T cell population. All immune studies were performed prior to the terminal phase of her illness and before she received blood transfusions. Further experiments of suppressor radiation sensitivity and the analysis of T cell helper function were not possible because the patient became se- verely ill and received a number of blood transfusions.

In vitro bone resorption assays were performed to deter-

OSTEOPOROSIS AND IMMUNOREGULATION 513

FIG. 2. osteoid surface is sometimes active (open arrow) but most often thin and lined with flat cells (straight arrows).

High magnification of a trabecula. Numerous small osteoclasts (curved arrows) and lacunae are present; the

URINARY CALCIUM TO CREATINE RATIO

- - 0 0 0 VI I I

CALCITONIN DOSAGE ( U )

0 vl

FIG. 3. Patient’s response to intramuscular salmon calcitonin. The top panel indicates dose of calcitonin adminis- tered and the bottom panel, the urinary calcium to creatinine ratio prior to and during the treatment trial.

mine whether the patient’s sera or mononuclear cells secreted a factor that activated osteoclasts. Three assays on two different serum samples at 10 and 20% concentrations showed no significant stimulation of bone resorption. Supernatants of PHA-stimulated peripheral mononuclear cell cultures showed significant stimulation of bone resorption at a 1:8 dilution compared to control BGJ medium but did not differ significantly from an age-matched sick control or from bone-resorbing activity usually found in PHA-slim- ulated supernatants of adults.

DISCUSSION

This patient had two distinct processes that contributed to her eventual demise. She had significant bone disease as evi- denced by the extensive bone erosions of the inner ear, osteopenia, and multiple pathologic fractures. In addition to her bone problem, she had recurrent pyogenic infections and underlying immune deficiency disorder that was diffi- cult to categorize into any of the known immune deficiency syndromes. She clearly had abnormalities of immunoregulation with increased T cell suppressor activity that affected her humoral immune B cell system. Although the serum immunoglobulin levels were normal, she had a markedly im-

514

TABLE 1. PWM-INDUCED B CELL IMMUNOGLOBULIN SYNTHESIS IN TISSUE CULTURE

RUBIN ET AL.

~

4 s kM &A ( w / m l ) (WmO ( W m l )

Patient 238a 188 180 (23-482)b (27-456)

Control 5883c 7798 4846 513 72 1 810

Yo Change - 63d - 87 - 82 (-12.5 to -91)b (-41 to -100) (-53 to -100)

aAverage of five testings over a 1 year period. bRange of values for the five testings. CMean f SEM of 38 subjects. dCoculture of patient’s mononuclear cells with control mononuclear cells. Nega-

tive values suggest increase suppressor activity. Values represent the mean of five testings.

TABLE 2. PWM-INDUCED IMMUNOGLOBULIN PRODUCTION WITH COMIXTURES OF T- AND B-ENRICHED

LYMPHOCYTE POPULATIONS ~ ~ ~

IgG IgM IgA Cells a (ng/ml) ( W m l ) ( W m l )

Bc + Tcb 2321 3495 5940 BP + TP 99 177 324 Bp + Tc 1724 5329 3869 Bc + Tp 370 177 313

aTc and Bc, control T cells and control B cells. Tp and Bp, pa-

bA 2:1 ratio of T cells to B cells or 1 x 10’ T cells in culture with tient T cells and patient B cells.

5 x lo4 B cells.

paired ability to produce functional antibodies that was related to her T cell abnormalities of immunoregulation. The major objective of our studies was to explore the possible interactions between the metabolic bone disease and her immune dysfunction.

At the time of the patient’s initial bone evaluation, the diagnosis of a form of severe osteogenesis imperfecta (01) was entertained. Besides the lack of a dominant family history and normal fibroblast typing (the ratio of collagen type 111 to I from the culture skin fibroblasts of patients with mild 0 1 is often increased),(”) the bone histomorphometry results showing numerous small osteoclastic cells led us to ex- clude the diagnosis of 01.

Another main area of differential diagnosis was that of a defect in mineralization, in particular a form of rickets with secondary hyperparathyroidism in which osteopenia and fractures can be a prominent feature. However, there was no radiographic evidence of rickets and/or osteoma-

lacia or secondary hyperparathyroidism, and her initial bone biopsy showed what appeared to be normal mineralization. Since the patient was receiving total parenteral nutrition (TPN) at the time of the initial bone evaluation, we considered the possibility of TPN-associated metabolic bone disease. ( l o ) However, our patient’s diffuse osteopenia and recurrent fractures were already apparent prior to the onset of her course of hyperalimentation. It is possible that the TPN may have exacerbated the underlying bone disease. Similarly, anticonvulsant therapy,(zzl nutritional factors, ( 2 3 1 and recurrent infections were likely to contribute to the severity of the underlying bone disease.

The result of the patient’s initial iliac crest biopsy en- abled us to classify her bone disease as osteoporosis with excessive bone resorption. Furthermore, the biochemical findings of a slightly increased serum free calcium, high urinary cyclic AMP, and normal immunoreactive PTH were suggestive of a nonparathyroid humoral factor that biologically resembles the activity of PTH. Studies of these parameters in patients with hypercalcemia of malignancy show a constellation of findings similar to that found in our patient. In this disorder, a PTH-related peptide (PTHrP) presumably is secreted by a solid This syndrome can be distinguished from classic hyperparathyroidism since normal immunoreactive PTH levels are found and increased osteoclastic resorption occurs without compensatory increased osteoblastic activity. Although no malignancy was found in our patient, her biochemistry and biopsy findings strongly suggested the presence of a factor that had activated osteoclasts resulting in a net bone loss.

In an attempt to identify a circulating resorptive factor, an in vitro bone resorption assay was performed that on three occasions failed to document increased resorptive activity in the patient’s serum or cell supernatant. The failure to identify a serum factor or lymphokine by our in vitro

OSTEOPOROSIS AND IMMU NOHEGULATION

assay system does not rule out the presence of such a factor in vibo. This could be due to insufficient culture condi- tions or to the presence of serum inhibitors of one or more bone resorbing factors. Furthermore, local bone-derived factors, which can regulate bone remodeling, may not be detected in this assay.‘zss’

Minor bone abnormalities have been observed in patients with certain types of immunodeficiency. For exam- ple, a chondro-osseous dysplasia has been described in adenosine deaminase deficiency (ADA) with severe combined immunodeficiency (SCID)(”6’ and metaphyseal changes have been observed in some patients with cartilage hair hypoplasia (CHH).[27’ In contrast to ADA with K I D and CHH, this patient had a more restrictive immunoregulatory defect of increased suppressor T cell function. Her bone disease was much more severe and generalized and not restricted, as in the above immunodeficiency disorders, to metaphyseal sites. It is possible that the various bone abnormalities that may occur in immunologically deficient individuals represent a spectrum of biochemical abnormalities, the most severe of which is illustrated by our case.

This patient’s malignant osteoporosis was an extraordi- nary finding, which was associated with an isolated abnormality in immunoregulation. Although this case may represent the coincidence of these two disorders in a single in- dividual, we speculate that these defects are linked by a common pathway. Candidate factors include interleukin-1 and tumor necrosis factor, which are two potent leuko- cyte-derived stimulators of bone resorption in hematologic malignancy, inflammation, and injury.tZ8 2 9 ) However, IL-1 and TNF have not been shown to increase urinary cyclic AMP production.

A PTHdike factor in the humoral hypercalcemia of malignancy (HHM), now termed PTH-related peptide (PTHrP). may be produced by epithelial cells. It has been shown to stimulate adenylate cyclase production, increase bone resorption, and inhibit bone formation. (30-33s’ It is possible that the production of an as-yet-unidentified cyto- kine, with properties similar to those of PTHrP, resulted in an interaction between bone and bone marrow cells that led to the enhanced bone resorption and increased suppressor activity found in this patient. Continued research in identifying the various factors, many of them immune cell derived, that regulate bone resorption will lead to a better understanding of patients with findings similar to those presented in this case and ultimately to successful therapeutic intervention.

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Address reprint requests to: Karen Rubin, M.D.

Department of Pediatrics University of Connecticut Health Center

263 Farmington Avenue Farmington, CT 06032

GR 1986 Stimulation of bone resorption and inhibition of bone formation in vitro by human tumor necrosis factors. Nature 3195 16-5 18.

Received for publication September 21, 1987; in revised form February 3, 1988; accepted May 31, 1988.

malignant osteoporosis and defective immunoregulation

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