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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To determine whether parathyroid hormone–related protein (PTHrP), an interleukin-1β–inducible, bone-resorbing peptide that is produced in increasing amounts by the synovium in rheumatoid arthritis (RA), may play a role in the pathophysiology of joint destruction in RA.

Methods

PTHrP expression and the effect of PTHrP 1–34 neutralizing antibody on disease progression were tested in streptococcal cell wall (SCW)–induced arthritis, an animal model of RA.

Results

As has been reported in RA, while serum levels of PTHrP did not change during SCW-induced arthritis, PTHrP expression dramatically increased in the arthritic synovium. Treatment with PTHrP neutralizing antibody (versus control antibody) did not affect joint swelling in SCW-treated animals. However, PTHrP antibody significantly inhibited SCW-induced joint destruction, as measured by its ability to block increases in serum pyridinoline (a marker of cartilage and bone destruction), erosion of articular cartilage, decreases in femoral bone mineral density, and increases in the numbers of osteoclasts in eroded bone. Unexpectedly, granuloma formation at sites of SCW deposition in the liver and spleen was also inhibited by PTHrP antibody, an effect associated with significant decreases in the tissue influx of PTH/PTHrP receptor–positive neutrophils and in SCW-induced neutrophilia. In vitro, neutrophil chemotaxis was stimulated by PTHrP 1–34.

Conclusion

These findings suggest that PTHrP, consistent with its previously described osteolytic effects in metastatic bone disease, can also be an important mediator of joint destruction in inflammatory bone disorders, such as RA. Moreover, this study reveals heretofore unknown effects of PTHrP peptides on neutrophil function that could have important implications in the pathogenesis of inflammatory granulomatous disorders.

In rheumatoid arthritis (RA), periarticular destruction of cartilage and bone occurs in response to local invasion of the joint by an expanding tumor-like rheumatoid synovium (1, 2). Inflammatory cytokines produced by the invading synovium, such as tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and IL-6, are primary mediators of joint inflammation and articular destruction in RA (1, 2). Indeed, inhibition of cytokine action has proved to be an effective target for new methods of therapeutic modalities, as evidenced by the successful use of TNFα blocking agents in the prevention of joint inflammation in RA (1, 2).

Parathyroid hormone–related protein (PTHrP) is a peptide which, like TNFα, was first identified because of its biologic effects in the setting of malignancy. When produced in prodigious amounts by tumors, PTHrP, by virtue of its ability to bind to and activate the G protein–coupled PTH/PTHrP receptor in bone, is the humoral factor responsible for marked bone resorption and hypercalcemia in this setting (3). However, in the absence of malignancy, PTHrP is produced in many organs, where it acts locally, rather than systemically (3). For example, in response to inflammatory stimuli, PTHrP expression has been demonstrated to increase in the liver, where it activates the hepatic acute-phase response (4).

More recently, PTHrP has also been identified as a member of the cascade of cytokines produced in prodigious amounts by the rheumatoid synovium (5, 6). As with other cytokines, PTHrP is expressed by synoviocytes, the primary cell comprising the tumor-like rheumatoid synovium (5–9). TNFα and IL-β, 2 key inflammatory cytokines in RA, induce synoviocyte PTHrP expression, and PTHrP, in turn, induces synoviocyte secretion of IL-6 (5). IL-6, a cytokine that mediates joint destruction and inflammation in animal models of arthritis (10), is also a critical mediator of PTH/PTHrP peptide–induced bone resorption (11, 12). Moreover, continuous exposure of bone to PTH/PTHrP peptides is a potent stimulus for TRANCE/receptor activator of nuclear factor κB (RANK)–mediated osteoclastic bone resorption (13), a pathway previously demonstrated to mediate destruction of cartilage and bone in arthritis (14). In addition, studies demonstrating PTH/PTHrP inhibition of chondrocyte collagen synthesis and extracellular inorganic pyrophosphate secretion (15–17) have identified PTH/PTHrP peptide–mediated pathways that might also contribute to cartilage destruction. These facts led us to postulate that the local increase in synovial PTHrP expression in RA may be a critical mediator of bone and cartilage destruction and that, by virtue of its induction by TNFα and its ability to induce IL-6 secretion, PTHrP may also contribute to joint inflammation in this setting.

To test these hypotheses, we used a well-described animal model of RA, streptococcal cell wall (SCW)–induced arthritis in female Lewis rats (18). In this model, intraperitoneal administration of purified Streptococcus group A–derived peptidoglycan polysaccharide results in the preferential deposition of these complexes in joints, liver, and spleen (18–21). Within the joint, SCW initiates an inflammatory response that ultimately leads to synovial tissue proliferation and joint erosion by invading pannus, a histopathologic lesion very similar to that seen in RA (18–21). Moreover, the inflammatory cytokines and T cells that mediate joint destruction in RA are similarly responsible for joint destruction in SCW arthritis (20–23). Inflammatory cytokines, such as TNFα, are also involved in the granulomatous response that develops in the liver and spleen in response to SCW deposition (24, 25). Initial experiments were therefore undertaken to determine whether PTHrP, analogous to its inducible expression in the human rheumatoid synovium, was also expressed by the inflammatory synovium in SCW arthritis. After establishing the suitability of this model for testing the role of endogenous PTHrP production in joint destruction, treatment trials using a PTHrP 1–34 neutralizing antibody were then initiated to determine whether joint inflammation and/or joint destruction could be prevented by blocking the effects of PTHrP. As a secondary outcome, the effects of PTHrP antibody treatment on the inflammatory granulomatous response in liver and spleen were also evaluated.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Animal procedures.

Following standard protocols (19–22) conducted in accordance with institutional guidelines, female Lewis rats (Harlan, Indianapolis, IN) were administered a single intraperitoneal injection of vehicle (normal saline) or peptidoglycan–polysaccharide polymers (25 μg rhamnose/gm body weight) isolated from the sonicated cell wall of group A Streptococcus pyogenes (Lee Laboratories, Grayson, GA). As previously described (21), following a transient decrease in weight during the first 24 hours, SCW-treated animals gained weight throughout the course of the experiment in parallel with controls and continued to walk and bear weight. One hour prior to SCW treatment, animals received an intraperitoneal injection of either control antibody or PTHrP neutralizing antibody. Antibody treatment continued twice weekly throughout the course of the experiment at a dose of either 3.0 μl/gm (at the start of treatment and on day 17 at the start of the chronic phase) or 1.5 μl/gm (all other times). Joint inflammation was determined in a blinded manner by daily assessment of arthritis index (AI) in each distal limb using standard criteria (20–22) (0 = normal, 1 = slight erythema and edema, 2 = increased edema with loss of landmarks, 3 = marked edema, 4 = marked edema with ankylosis on flexion). Reproducibility of AI assessment was verified in initial experiments (SMD, GS) by correlation with caliper measurements of joint swelling.

PTHrP neutralizing antibody preparation.

Antiserum was generated against synthetic rat PTHrP 1–34 (Bachem, Torrance, CA) by immunization of goats according to standard techniques (Covance, Berkeley, CA). The immunoglobulin fraction of immune (PTHrP antibody) or naive (control antibody) serum was partially purified by ammonium sulfate precipitation using apyrogenic techniques (26). Endotoxin content of the antibody solutions (≤36 pg/dose) was determined by Limulus assay (27), and antibody titer (1:30,000) was determined by enzyme-lined immunosorbent assay. The specific ability of PTHrP antibody to neutralize PTHrP 1–34 bioactivity was verified in vitro by assessment of the effect of antibody treatment on rat PTHrP 1–34 versus rat PTH 1–34 (Bachem) stimulation of cAMP formation in a rat UMR-106 osteosarcoma cell line (American Type Culture Collection, Manassas, VA) using previously described techniques (28). Briefly, prior to a 15-minute incubation with UMR-106 cells (1 × 105/well in a 24-well plate), 10−8M PTHrP 1–34 and 10−8M PTH 1–34 peptide solutions, diluted in Dulbecco's modified Eagle's medium and 10% fetal calf serum, were preincubated for 2 hours at 37°C with the indicated dilution of PTHrP antibody, control antibody, or media alone. The cAMP content of cell lysates was determined by competitive immunoassay (Correlate-EIA direct cAMP assay; Assay Designs, Ann Arbor, MI).

PTHrP, osteocalcin, and pyridinoline assays.

Plasma, collected in the presence of protease inhibitors, was tested for PTHrP using a 2-site immunoradiometric assay (IRMA) (sensitivity 0.3 pM) using antibodies directed against PTHrP 1–40 and PTHrP 60–72 (Nichols Institute Diagnostics, San Juan Capistrano, CA). Plasma levels of osteocalcin, a marker of bone formation, were determined using a commercially available IRMA specific for rats (Immutopics, San Clemente, CA). A competitive enzyme immunoassay (Metra Biosystems, Mountain View, CA) was used to evaluate pyridinoline, a marker of cartilage and bone destruction (29–32).

Histology.

Tissue specimens were fixed in 10% formalin, and joints were subsequently decalcified in 10% EDTA (pH 7.0) and tissues were embedded in paraffin. Tissue sections were processed for immunohistochemical staining as previously described (5) using affinity-purified polyclonal primary antibodies directed against PTHrP 34–53 (Oncogene Science, Cambridge, MA) or the rat PTH/PTHrP receptor (amino acids 90–106) (a kind gift from Dr. Robert Nissenson, University of California at San Francisco). Liver and spleen sections were processed for antigen unmasking by heating for 10 minutes at 95°C in 10 mM sodium citrate (pH 6.0) prior to immunostaining. In all tissues, specificity of PTHrP or PTH/PTHrP receptor immunoreactivity was verified by the absence of staining that resulted when sequential sections were treated with antibodies that had been preincubated with a 30- to 20-fold excess by weight of their respective antigens (obtained from Oncogene Science and Dr. Robert Nissenson, respectively), as well as with a nonspecific control IgG. All histologic analyses were performed in a blinded manner.

Osteoclasts were identified by tartrate-resistant acid phosphatase staining (Sigma, St. Louis, MO) and counted in the area of the distal tibial growth plate at the site of pannus invasion in the hind limbs 28–34 days after SCW (or vehicle) injection (14). An index of articular cartilage destruction (20, 21) was calculated by assessment of articular cartilage in the distal tibia in hematoxylin and eosin (H&E)–stained sections of hind joints obtained 28–34 days after SCW (or vehicle) injection (0 = normal, 1 = minimal destruction, 2 = at least 50% destroyed, 3 = entirely destroyed). Use of H&E-stained joints for cartilage assessment was verified in parallel studies using a subset of joints stained with toluidine blue to visualize the proteoglycan matrix (14), which was not destroyed in SCW arthritis in the absence of invasion of the articular cartilage by synovial tissue. Standard histologic parameters of synovitis (pannus formation and inflammatory cell infiltration) and periarticular bone destruction were also assessed in H&E-stained sections of hind joints (10, 14, 19, 20). Granuloma formation was assessed in H&E-stained sections of liver and spleen using standard criteria (19–21). Neutrophils were identified in liver and spleen sections by naphthol-AS-D chloroacetate esterase staining (Sigma) (33).

Bone mineral density.

Bone mineral density (BMD) of the distal 25% of the hind femur was determined using a Piximus densitometer (General Electric Medical Systems, Milwaukee, WI) during the established chronic phase of arthritis (day 33) in SCW-injected animals treated with control antibody versus PTHrP antibody, or in normal animals receiving vehicle alone. The distal femur was chosen to assess the effects of PTHrP antibody treatment on BMD, since this cancellous site was adjacent to an involved joint and demonstrated a marked loss of trabecular bone on histologic assessment in association with a marked decrease in BMD in SCW-treated animals.

White blood cell counts.

Circulating white blood cell counts were determined by automated analysis using a Hemavet 850 analyzer (Drew Scientific, Oxford, CT). Differential cell counts were determined in a blinded manner by manual counting using standard methods.

Neutrophil chemotaxis.

Elicited peritoneal neutrophils were isolated from female Lewis rats 12 hours after intraperitoneal injection of 3 cc sterile Difco NIH thioglycollate broth (Becton Dickinson, Sparks, MD) (34). Subsequent to erythrocyte lysis (WL1000; R&D Systems, Minneapolis, MN), neutrophils, identified by differential counting of stained cells (Hemacolor; EMD, Gibbstown, NJ), were plated at 5 × 104 cells/well in Hanks' balanced salt solution (HBSS) in the upper chamber of a 48-well Boyden chamber (35). Lower chambers were loaded with HBSS containing 0.1% bovine serum albumin and the indicated concentrations of PTHrP 1–34, or with 1 × 10−6M FMLP (Sigma) as a positive chemotaxis control (35). The number of neutrophils migrating through the intervening 3-μm pore size, polyvinylpyrrolidone-free, polycarbonate membrane (Osmonics, Minnetonka, MN) was determined after 40 minutes of incubation at 37°C by counting neutrophils adhering to the bottom of the Hemacolor-stained membrane (35, 36).

Statistical analysis.

Values are presented as the mean ± SEM, with statistical significance determined by analysis of variance with post hoc testing or by Fisher's exact test, as appropriate, using InStat software (GraphPad Software, San Diego, CA).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

PTHrP expression in SCW-induced arthritis.

Circulating plasma levels of PTHrP 1–72 were unchanged in SCW-treated versus control animals during the peak phase of acute arthritis (control 0.64 ± 0.03 pM versus SCW 0.63 ± 0.04 pM) or chronic arthritis (control 0.52 ± 0.07 pM versus SCW 0.47 ± 0.04 pM). In contrast, local PTHrP expression increased dramatically in arthritic joints. In normal joints (Figure 1A), PTHrP is primarily located in articular chondrocytes and synoviocytes that form the delicate lining of normal synovium. In SCW-treated animals (Figures 1B, E, and G), analogous to the histopathologic changes seen in RA, the synovium grows in a tumor-like manner, filling the joint space and invading cartilage and bone during the chronic phase of arthritis. Immunoreactive PTHrP was located throughout the inflammatory synovium, including tissue directly invading articular cartilage and occupying marrow cavities (Figures 1B and E). As in RA (5, 7, 8), synoviocytes and the synovial vasculature, the sites of cytokine production in RA, were PTHrP-positive in the SCW synovium (Figures 1B, C, and E). Specificity of PTHrP staining in all cases was verified by the absence of staining documented in consecutive sections incubated with PTHrP antibody that had been preincubated with an excess of antigen, as well as the absence of staining documented with nonspecific rabbit IgG (Figures 1D and F).

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Figure 1. Histologic assessment of streptococcal cell wall (SCW) inflammation. A, Immunohistochemical staining for parathyroid hormone–related protein (PTHrP) (brown) in a normal joint is present in the synovial tissue (st) lining cells, as well as articular chondrocytes (ac). B, Immunohistochemical staining for PTHrP in a joint from an animal with established chronic arthritis is present throughout the synovium in synoviocytes and synovial vasculature, including synovial tissue invading cartilage and bone (arrows). C, In this higher-magnification view, immunoreactive PTHrP can be seen in synoviocytes (see arrows for examples) that comprise the tumor-like synovium, as well as vascular cells. v = vessels. D, Specificity of synoviocyte (see arrows) and vascular PTHrP staining in C is demonstrated by comparison with the absence of staining seen on a consecutive section incubated with a nonspecific IgG. E, Synovial tissue invading periarticular marrow cavities is also PTHrP positive, as can be seen in this tibia, where the epiphyseal cartilage (ec) is being destroyed. tb = trabecular bone. F, Specificity of immunoreactive PTHrP in invading synovial tissue in E is demonstrated by comparison with the absence of staining on a consecutive section where PTHrP antibody was preincubated with an excess of antigen. G, Tibial cartilage articular chondrocytes and epiphyseal bone and marrow are invaded by synovial tissue in a joint from SCW-animal treated with control antibody. H, Tibial cartilage articular chondrocytes and epiphyseal bone (pink) and marrow (m) are relatively preserved in SCW animal treated with PTHrP antibody, despite abnormal overgrowth of synovial tissue in the joint space and the same clinical arthritis index as the joint shown in G. Bar = 100 μm in A, E, and G; 30 μm in C.

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PTHrP antibody specifically neutralizes PTHrP 1–34 bioactivity.

PTHrP antibody specifically inhibited PTHrP 1–34 stimulation of cAMP (Figure 2) in rat osteosarcoma cells, while having no effect on cAMP stimulation by PTH 1–34 (Figure 2), a peptide that also activates the PTH/PTHrP receptor. The median effective dose for inhibition of cAMP formation stimulated by 10−8M PTHrP, a PTHrP concentration 10,000-fold higher than that found in rat plasma, was achieved with an antibody dilution of 1:140 (data not shown). PTHrP antibody doses used for in vivo experiments (1.5–3 μl of 1:30,000 titer antibody/gm body weight) were the same as those previously shown to prevent lethality in mice during endotoxemia (26), an amount sufficient to neutralize 30–60 pmoles of PTHrP 1–34. Twice weekly antibody dosing was based on other successful antibody treatment protocols used for cytokine blockade studies in rodent arthritis models (37, 38).

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Figure 2. Effect of parathyroid hormone–related protein 1–34 (PTHrP 1–34) neutralizing antibody (AB) on PTHrP 1–34– or PTH 1–34–stimulated cAMP formation. UMR-106 cells were treated for 15 minutes with 10−8M PTHrP 1–34 or 10−8M PTH 1–34 in the absence or presence of PTHrP neutralizing antibody or control antibody (diluted 1:100). Accumulated cAMP, measured in the supernatants of the lysed cells, as described in Materials and Methods, is expressed as the mean ± SEM (n = 4 wells/condition). The cAMP levels in untreated cells (1.3 ± 0.2 pmoles/ml) were unchanged by antibody treatment alone. ∗∗∗ = P < 0.001 versus PTHrP 1–34 alone, by analysis of variance.

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Effect of PTHrP antibody treatment on joint inflammation.

As previously described (19–22), SCW-treated animals developed an acute phase of joint inflammation that peaked on days 3–5, subsided by days 10–12, and was then followed by a chronic phase of arthritis that was associated with actual joint destruction (Figure 3, open circles). Treatment of animals twice per week with PTHrP neutralizing antibody, beginning 1 hour prior to SCW administration, had no effect on the development of either acute or chronic joint inflammation (Figure 3, solid circles). This lack of effect of PTHrP neutralizing antibody (versus control antibody) on joint inflammation was verified in multiple experiments by AI and by histologic assessment of synovitis (data not shown).

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Figure 3. Effect of PTHrP neutralizing antibody on the arthritis index. Female Lewis rats were injected on day 0 with streptococcal cell wall (SCW; 25 μg/gm) 1 hour after intraperitoneal treatment with control or PTHrP antibody. Antibody treatment was continued twice weekly throughout the course of the experiment. Joint swelling was assessed by daily calculation of the arthritis index in this representative experiment, as described in Materials and Methods. Values are the mean ± SEM of 7–9 animals/group. There were no statistically significant differences between the treatment groups at any time point. See Figure 2 for other definitions.

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Effect of PTHrP antibody treatment on joint destruction.

Periarticular bone loss has been reported to occur in association with histologically evident joint destruction during the chronic phase of SCW-induced arthritis (19–21, 39). To further characterize joint destruction and bone loss in this model, serum markers of bone formation (osteocalcin) and of bone resorption and cartilage destruction (pyridinoline) were measured. Serum osteocalcin levels were unchanged during the established chronic phase of arthritis (control 55.6 ± 3.6 ng/ml versus SCW 56.2 ± 6.1 ng/ml) and remained unchanged with PTHrP antibody treatment (data not shown). In contrast, serum pyridinoline levels increased almost 2-fold during the chronic phase of SCW arthritis (Figure 4A). Treatment with PTHrP antibody inhibited the SCW-induced increase in pyridinoline, a degradation product of cartilage and bone that correlates with destruction of both of these tissues in animal models of arthritis (29–32) (Figure 4A).

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Figure 4. Effect of PTHrP neutralizing antibody on cartilage and bone destruction. A, Blood was drawn from the inferior vena cava of anesthetized rats that received vehicle alone, or streptococcal cell wall (SCW)–injected animals treated with control or PTHrP antibody 28–34 days after SCW or vehicle administration. Serum pyridinoline levels, assayed as described in Materials and Methods, are expressed as the mean and SEM (n = 9–15/group). The baseline pyridinoline level in normal control animals was 4.3 ± 0.2 nM. B, Destruction of articular cartilage, assessed as described in Materials and Methods, was determined in joints obtained from rats receiving vehicle alone, or SCW-injected animals treated with control or PTHrP antibody 28–34 days after SCW (or vehicle) administration. Index of destruction (0–3 scale) is expressed as the mean and SEM (n = 18–20/group). C, Bone mineral density (BMD) of the distal femur of the hind legs was determined in rats receiving vehicle alone, or SCW-injected animals treated with control or PTHrP antibody 33 days after SCW (or vehicle) administration. BMD is expressed as the mean and SEM (n = 8–12/group). D, Tartrate-resistant acid phosphatase–positive osteoclasts were identified, as described in Materials and Methods, in the distal tibia of hind legs obtained from rats receiving vehicle alone, or SCW-injected animals treated with control or PTHrP antibody 34 days after SCW (or vehicle) administration. The number of osteoclasts (NOC) per area of tibia at the site of pannus invasion is expressed as the mean and SEM (n = 3–6/group). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 (the 2 experimental conditions versus each other as indicated, or the experimental condition versus vehicle treatment alone), by analysis of variance. See Figure 2 for other definitions.

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Histologic evaluation of articular cartilage destruction during the established phase of chronic arthritis in joints from animals treated with control antibody (Figure 1C) versus PTHrP antibody (Figure 1D) revealed a relative preservation of articular cartilage in those animals receiving PTHrP antibody (Figure 4B). PTHrP antibody treatment also prevented 30% of the decrease in femoral BMD that occurred with established arthritis (Figure 4C). The 8-fold increase in the number of bone-resorbing osteoclasts at the site of pannus invasion into the tibia of arthritic, control antibody–treated animals was also significantly inhibited (−40%) by PTHrP antibody treatment (Figure 4D). Consistent with these results, histologic assessment of bone destruction also demonstrated a relative preservation of trabecular bone in the proximal tibia (Figures 5A–C) and proximal femur (Figures 5D–F) of animals treated with PTHrP antibody, despite continued inflammation.

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Figure 5. Effect of parathyroid hormone–related protein (PTHrP) neutralization on periarticular bone histology. A, Distal tibia from a normal control animal, demonstrating normal bone (pink), epiphyseal growth plate (ep), and marrow space (m), as well as normal synovial tissue (st). B, In a typical distal tibia from a streptococcal cell wall (SCW)–treated animal, synovial tissue grows in a tumor-like manner, in which the epiphyseal growth plate and trabecular bone in epiphysis and metaphysis have been destroyed, and marrow is filled with hemopoietic cells (purple). C, In a typical distal tibia from an SCW-treated animal treated with PTHrP antibody, despite a similar degree of synovitis, trabecular bone (pink) and epiphyseal plate are relatively preserved. D, Normal trabecular bone (see arrow for example) in the epiphysis (epiph) and metaphysis (meta) of the distal femur from a control animal. E, Trabecular bone (arrow) is decreased in the epiphysis and metaphysis of the distal femur from an SCW-treated animal. F, PTHrP antibody treatment protects trabecular bone (arrow) in the epiphysis and metaphysis of the distal femur from an SCW-treated animal.

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Effect of PTHrP antibody treatment on hepatic and splenic granulomas.

As has been previously reported (18–22), the majority of animals with SCW-induced arthritis developed granulomas in their livers and spleens (Figure 6, open bars). However, treatment with PTHrP antibody significantly decreased the incidence of hepatic and splenic granuloma formation in SCW-treated animals (−36% and −62%, respectively) (Figure 6, hatched bars). Moreover, in the subset of PTHrP antibody–treated animals that still had some evidence of granuloma formation, the number and size of the granulomas were also reduced. For example, PTHrP antibody–treated animals with hepatic granulomas had one-third the number of lesions and no evidence of large macrogranulomas as defined by Geratz et al (19) (versus 64% hepatic macrogranulomas [percent of total granulomas] in control antibody–treated animals, P < 0.0001).

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Figure 6. Effect of PTHrP neutralizing antibody on granuloma formation. Granulomas were identified by histologic analysis of hematoxylin and eosin–stained sections of liver and spleen harvested 28–34 days after streptococcal cell wall injection in control (Con) or PTHrP antibody–treated Lewis rats (n = 12–21 animals/treatment group). ∗ = P < 0.05; ∗∗ = P = < 0.01, by Fisher's exact test. See Figure 2 for other definitions.

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Effect of PTHrP antibody treatment on neutrophil influx into the liver and spleen.

PTHrP was expressed in the granulomatous lesions of the liver (Figures 7A and B) and spleen (not shown) by epithelioid cells inside the fibrotic margins of the lesions and by the vasculature, while the PTH/PTHrP receptor was primarily expressed in multinucleated cells at the necrotic centers (Figures 7A and C). Staining of sequential sections of granulomatous livers and spleens for neutrophils and for the PTH/PTHrP receptor identified neutrophils as a site of PTH/PTHrP receptor expression in the granulomas (Figures 7D and E), normal parenchyma (Figures 7F and G), and vascular lumens (Figures 7H and I). The dramatic increase in the number of neutrophils within the parenchyma of the liver and spleen in SCW animals (Figure 8A) was markedly inhibited by PTHrP antibody treatment (−48% and −88%, respectively) (Figure 8A). Of note, the 2-fold greater inhibitory effect of PTHrP antibody on neutrophil accumulation in the spleen (versus the liver) (Figure 8A) paralleled its 2-fold greater inhibitory effect on granuloma formation in this organ (Figure 6). In the perivascular areas of the liver, where neutrophils are localized in early inflammation (19), PTHrP antibody treatment also inhibited neutrophil influx (52% inhibition; P < 0.001).

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Figure 7. Localization of immunoreactive parathyroid hormone–related protein (PTHrP), PTH/PTHrP receptor, and neutrophils in granulomas. A, Multinucleated cells (mn) in the necrotic centers of hematoxylin and eosin–stained hepatic granulomas (GR) are surrounded by epithelioid cells (ep), while the margin is composed of fibroblast-like cells (fb). NL = normal hepatocytes. B, Immunoreactive PTHrP staining (brown) in a consecutive section of the hepatic granuloma seen in A is primarily located in epithelioid cells and in the vasculature (arrow). Normal hepatocytes are also PTHrP positive, as previously described (4). C, In contrast, within the granuloma, the PTH/PTHrP receptor (brown) is mainly located in multinucleated cells in the granuloma center. D, Multinucleated cells in the center of a hepatic granuloma are neutrophils, as determined by naphthol-AS-D chloroacetate esterase staining (pink). E, Neutrophils in a consecutive section of the hepatic granuloma seen in D are immunoreactive for the PTH/PTHrP receptor (brown). F, Increased numbers of naphthol-AS-D chloroacetate esterase–positive neutrophils (pink) are present in the red pulp of a granulomatous spleen. G, Multinucleated cells in the same area of spleen shown in F are immunoreactive for the PTH/PTHrP receptor (brown). H, Naphthol-AS-D chloroacetate esterase–positive neutrophils (pink) are marginated in the hepatic vein of a portal triad (see arrow for example). I, Marginated neutrophils in a consecutive section of the portal triad seen in H are immunoreactive for PTH/PTHrP receptor (brown). Nuclei are counterstained with methyl green.

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Figure 8. Effects of PTHrP 1–34 neutralizing antibody or PTHrP 1–34 peptide on neutrophils. A, Naphthol-AS-D chloroacetate esterase–positive neutrophils in the liver parenchyma (excluding marginated cells in vessels) or splenic red pulp, expressed as the number/mm2 (n = 4–9 animals/group), was ascertained in a blinded manner in control animals receiving vehicle alone or in streptococcal cell wall (SCW)–injected animals (day 29 postinjection) treated with control or PTHrP antibody. B, Chemotaxis of neutrophils toward the indicated concentrations of PTHrP 1–34 or of 1 × 10−6M FMLP, determined as described in Materials and Methods, expressed as the number of chemotaxed neutrophils/mm2 of membrane (n = 6–18 wells/treatment). Results are representative of 4 separate experiments. C, Concentration of circulating neutrophils, determined in blood obtained from control animals or SCW-injected animals treated with control or PTHrP antibody 35 days after injection of SCW (n = 4–6 animals/group). Values are the mean and SEM. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 (the 2 experimental conditions versus each other as indicated, or the experimental condition versus vehicle alone), by analysis of variance. See Figure 2 for other definitions.

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Effect of PTHrP 1–34 on neutrophil chemotaxis.

PTHrP 1–34 induced neutrophil chemotaxis in vitro (Figure 8B). Lower concentrations of PTHrP 1–34 (10−10M) were more potent stimulators of neutrophil chemotaxis than were higher (10−7M) concentrations (Figure 8B). The magnitude of the chemotaxic effect of PTHrP 1–34 was similar to that of 10−6M FMLP, an optimal dose of a known chemotaxic agent (Figure 8B, solid bar) (32).

Effect of PTHrP antibody on circulating neutrophils.

As has been previously reported (19), leukocytosis (control 5.2 ± 0.4 × 106 cells/ml versus SCW 9.0 ± 0.4 × 106 cells/ml; P < 0.001), with elevations in the number of circulating neutrophils, lymphocytes, and monocytes (data not shown), accompanied SCW inflammation. PTHrP antibody treatment partially inhibited SCW-induced leukocytosis (−43%; P < 0.05). This inhibitory effect was entirely due to the ability of PTHrP antibody to prevent SCW-induced neutrophilia (Figure 8C), because circulating numbers of lymphocytes and monocytes were not affected by PTHrP antibody treatment (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Two novel biologic effects of PTHrP are demonstrated in this study: PTHrP mediation of joint destruction in inflammatory arthritis, and PTHrP regulation of granuloma formation and neutrophil function. The first effect of PTHrP appears to be analogous to the well-described ability of PTHrP to mediate osteolysis in metastatic bone disease, while the second effect of PTHrP represents an entirely new aspect of PTHrP bioactivity that would not be easily predicted from our current understanding of this multifunctional hormone. Indeed, because neutrophils are believed to contribute to bone loss in other types of inflammation and to mediate cartilage destruction in RA (40, 41), it is possible that normalization of circulating neutrophil counts by PTHrP blockade may also have contributed to the protective effect of this treatment on cartilage and bone during inflammation.

The joint protective effects of PTHrP blockade in SCW arthritis are similar to those reported by Guise et al (42) for an animal model of breast metastases wherein PTHrP neutralizing antibody treatment prevented osteolytic bone destruction in a setting of localized, but not systemic, increases in PTHrP expression by invading tumor metastases. Similarly, in the SCW model, because the inflammatory pannus that invades cartilage and bone is composed of PTHrP-positive cells, while systemic PTHrP levels are not elevated, it would appear that locally produced PTHrP within the tumor-like, invading pannus mediates osteolytic bone resorption as well as cartilage destruction. Because the TRANCE/RANK pathway has previously been demonstrated to mediate the osteolytic bone loss occurring both in malignancy and arthritis, as well as PTH/PTHrP-peptide–dependent bone loss (13, 14), these findings suggest that PTHrP induction of TRANCE and the subsequent induction of osteoclastogenesis may be an important mechanism of bone loss in both of these disease states.

In contrast to its effect in preventing destruction of cartilage and bone, PTHrP blockade had no effect on joint inflammation. As with any negative finding, it is impossible to rule out insufficient antibody dosing or delivery as the cause of this negative result. However, PTHrP antibody treatment did prevent other aspects of SCW-induced joint pathology. Moreover, other similarly designed studies have demonstrated the ability of systemically administered antibodies to distribute to joints and to prevent joint inflammation (37, 38, 43). Therefore, these results strongly suggest that endogenous PTHrP production does not mediate joint inflammation.

The ability of PTHrP to mediate joint destruction while not affecting joint inflammation is similar to the reported effect of IL-1 in arthritis, since IL-1 blockade in animal arthritis models also predominantly prevents joint destruction rather than inflammation (44). Because IL-1 induces PTHrP expression in synoviocytes (5), it is therefore possible that PTHrP mediates some of the joint-destructive effects of IL-1 in inflammatory arthritis. Alternatively, because PTHrP has previously been shown to synergize with TNF or IL-1 in destroying bone (45, 46), it is also possible that the partially protective effect of PTHrP antibody treatment, which prevented 30–60% of all measures of bone or cartilage destruction, is due to synergistic or additive effects of PTHrP with other inflammatory cytokines, such as IL-1β or TNFα.

The ability of PTHrP blockade to prevent SCW-induced granuloma formation in both liver and spleen was an unanticipated finding in these studies. Because the granulomatous response serves a protective function in walling off foreign bodies, stimulation of granuloma formation by endogenous PTHrP should be inherently beneficial. Conversely, inhibition of granuloma formation could lead to increased morbidity. However, in this noninfectious model, liver function was normal in SCW animals with granulomatous livers and remained unchanged with PTHrP antibody treatment (data not shown). Growing evidence suggests that PTHrP may be involved in the pathogenesis of multiple types of granulomatous disorders, because its increased expression has now been documented in sarcoidosis, schistosomiasis, giant cell granulomas, and idiopathic systemic granulomatous disease (47–50). However, to our knowledge, the results presented here are the first to identify a function for PTHrP in this pathologic process.

Because neutrophils are an important component of SCW granuloma formation (19), the evidence presented here would suggest that inhibition of neutrophil accumulation in the liver and spleen contributes to the protective effect of PTHrP blockade. Indeed, because perivascular epithelioid cell accumulation precedes neutrophil influx and SCW granuloma formation in the liver and spleen (19, 20), we hypothesized that PTHrP expression by the vasculature and/or epithelioid cells during early inflammation could induce the chemotaxis of PTH/PTHrP receptor–positive neutrophils to sites of SCW deposition in these organs. Consistent with this hypothesis, PTHrP 1–34, the peptide neutralized in our in vivo SCW studies, stimulated the chemotaxis of neutrophils in vitro. To our knowledge, neither PTH/PTHrP receptor expression nor PTHrP 1–34–stimulated chemotaxis has previously been reported in neutrophils. However, G protein–coupled receptors have been identified as critical mediators of neutrophil chemotaxis (51), and chemotaxic effects of PTH/PTHrP 1–34 peptides have been demonstrated in other cell types (52, 53). Indeed, Halstead et al (53) have reported a maximal response of osteoblasts to the chemotaxic effect of low-dose (10−10M) PTH 1–34 that is similar to the concentration-dependent effects demonstrated here for PTHrP 1–34 and neutrophils.

The apparent decrease in neutrophil influx into the livers and spleens of animals treated with PTHrP antibody may also be related to the ability of PTHrP blockade to prevent SCW-induced neutrophilia. While the exact mechanisms contributing to neutrophilia in SCW arthritis are not known, previous demonstrations of PTH 1–34 stimulation of granulocyte-macrophage colony-stimulating factor production are consistent with possible stimulatory effects of PTHrP 1–34 on neutrophil production (54, 55).

PTHrP is a multifunctional peptide whose role in inflammation is only beginning to be understood (3, 56). Previous studies conducted in our laboratory suggest that its function in this setting is dependent on the type of inflammatory stimulus and the site of PTHrP induction (56, 57). Thus, just as induction of PTHrP in the liver during endotoxemia can stimulate the hepatic acute-phase response (4), or induction of PTHrP in the cerebrovasculature during ischemia may prevent neuronal death by enhancing local blood flow (57), induction of this bone-resorbing peptide within the joint during inflammatory arthritis may contribute to periarticular joint destruction and previously unreported effects of PTHrP on neutrophil function may contribute to local granuloma formation.

In conclusion, the data presented here demonstrate that PTHrP-mediated osteolytic bone destruction is not only an important component of metastatic bone disease (42, 58), but that localized PTHrP expression can also significantly contribute to joint destruction in inflammatory arthritis. Moreover, because inhibition of PTHrP 1–34 activity blocked degradation of cartilage and bone during established arthritis in this animal model, PTHrP may also be a potential target for therapeutic interventions in patients with RA. At the same time, a cautionary note must also be sounded in light of the other striking finding in this study: the critical role of PTHrP in regulating granuloma formation in both the liver and the spleen. Just as reports have documented an increase in disseminated tuberculosis in RA patients treated with TNFα neutralizing agents (59), this same risk could also accompany the use of PTHrP blocking agents. At the same time, the identification of novel PTHrP actions in regulating the granulomatous response and/or neutrophil function during inflammation also provides fresh insights into the multifunctional effects of PTH/PTHrP peptides as they become available for clinical use.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Choy EHS, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 2001; 12: 90716.
  • 2
    Maini RN, Taylor PC. Anti-cytokine therapy for rheumatoid arthritis. Annu Rev Med 2000; 51: 20729.
  • 3
    Philbrick WM, Wysolmerski JJ, Galbraith S, Holt E, Orloff JJ, Yang KH. Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol Rev 1996; 76: 12773.
  • 4
    Funk JL, Moser AH, Grunfeld C, Feingold KR. Parathyroid hormone-related protein is induced in the adult liver during endotoxemia and stimulates the hepatic acute phase response. Endocrinology 1997; 138: 266573.
  • 5
    Funk JL, Cordaro LA, Wei H, Benjamin JB, Yocum DE. Synovium as a source of increased amino-terminal parathyroid hormone-related protein expression in rheumatoid arthritis: a possible role for locally produced parathyroid hormone-related protein in the pathogenesis of rheumatoid arthritis. J Clin Invest 1998; 101: 136271.
  • 6
    Horiuchi T, Yoshida T, Koshihara Y, Sakamoto H, Kanai H, Yamamoto S, et al. The increase of parathyroid hormone-related protein and cytokine levels in synovial fluid of elderly rheumatoid arthritis and osteoarthritis. Endocr J 1999; 46: 6439.
  • 7
    Okano K, Tsukazaki T, Ohtsuru A, Namba H, Osaki M, Iwasaki K, et al. Parathyroid hormone-related protein in synovial fluid and disease activity of rheumatoid arthritis. Br J Rheumatol 1996; 35: 105662.
  • 8
    Kohno H, Shigeno C, Kasai R, Akiyama H, Iida H, Tsuboyama T, et al. Synovial fluids from patients with osteoarthritis and rheumatoid arthritis contain high levels of parathyroid hormone-related protein. J Bone Miner Res 1997; 12: 84754.
  • 9
    Romas E, Kartsogiannis V, Ho PWM, Mosely JM, Martin TJ. PTHrP is expressed in chronic inflammatory arthritis [abstract]. J Bone Miner Res 1997; 12: S513.
  • 10
    Alonzi T, Fattori E, Lazzaro D, Costa P, Probert L, Kollias G, et al. Interleukin 6 is required for the development of collagen-induced arthritis. J Exp Med 1998; 187: 4618.
  • 11
    Greenfield EM, Shaw SM, Gornik SA, Banks MA. Adenyl cyclase and interleukin 6 are downstream effectors of parathyroid hormone resulting in stimulation of bone resorption. J Clin Invest 1995; 96: 123844.
  • 12
    Grey A, Mitnick MA, Masiukiewicz U, Sun BH, Rudikoff S, Jilka RL, et al. A role for interleukin-6 in parathyroid hormone-induced bone resorption in vivo. Endocrinology 1999; 140: 468390.
  • 13
    Ma YL, Cain RL, Halladay DL, Yang X, Zeng Q, Miles RR, et al. Catabolic effects of continuous human PTH(1–38) in vivo is associated with sustained stimulation of RANKL and inhibition of osteoprotegerin and gene-associated bone formation. Endocrinology 2001; 142: 404754.
  • 14
    Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999; 402: 3049.
  • 15
    Terkeltaub R, Lotz M, Johnson K, Deng D, Hashimoto S, Goldring MB, et al. Parathyroid hormone–related protein is abundant in osteoarthritic cartilage, and the parathyroid hormone–related protein 1–173 isoform is selectively induced by transforming growth factor β in articular chondrocytes and suppresses generation of extracellular inorganic pyrophosphate. Arthritis Rheum 1998; 41: 215264.
  • 16
    O'Keefe RJ, Loveys LS, Hicks DG, Reynolds PR, Crabb ID, Puzas JE, et al. Differential regulation of type-II and type-X collagen synthesis by parathyroid hormone-related protein in chick growth-plate chondrocytes. J Orthop Res 1997; 15: 16274.
  • 17
    Tsukazaki T, Ohtsuru A, Namba H, Oda J, Motomura K, Osaki M, et al. Parathyroid hormone-related protein (PTHrP) action in rat articular chondrocytes: comparison of PTH(1-34), PTHrP(1-34), PTHrP(1-141), PTHrP(100-114) and antisense oligonucleotides against PTHrP. J Endocrinol 1996; 150: 35968.
  • 18
    Cromartie WJ, Craddock JG, Schwab JH, Anderle SK, Yang CH. Arthritis in rats after systemic injection of streptococcal cells or cell walls. J Exp Med 1977; 146: 15851602.
  • 19
    Geratz JD, Tidwell RR, Schwab JH, Anderle SK, Pryzwansky KB. Sequential events in the pathogenesis of streptococcal cell wall-induced arthritis and their modulation by bis(5-amidino-2-benzimidazolyl)methane (BABIM). Am J Pathol 1990; 136: 90921.
  • 20
    Allen JB, Malone DG, Wahl SM, Calandra GB, Wilder RL. Role of the thymus in streptococcal cell wall-induced arthritis and hepatic granuloma formation: comparative studies of pathology and cell wall distribution in athymic and euthymic rats. J Clin Invest 1985; 76: 104256.
  • 21
    Yocum DE, Allen JB, Wahl SM, Calandra GB, Wilder RL. Inhibition by cyclosporin A of streptococcal cell wall–induced arthritis and hepatic granulomas in rats. Arthritis Rheum 1986; 29: 26273.
  • 22
    Brandes ME, Allen JB, Ogawa Y, Wahl SM. Transforming growth factor-β1 suppresses acute and chronic arthritis in experimental animals. J Clin Invest 1991; 87: 110813.
  • 23
    Makarov SS, Olsen JC, Johnston WN, Anderle SK, Brown RR, Baldwin AS Jr, et al. Suppression of experimental arthritis by gene transfer of interleukin-1 receptor antagonist cDNA. Proc Natl Acad Sci U S A 1996; 93: 4026.
  • 24
    Wahl SM, Allen JB, Dougherty S, Evequoz V, Pluznik DH, Wilder RL, et al. T lymphocyte-dependent evolution of bacterial cell wall-induced hepatic granulomas. J Immunol 1986; 137: 2199209.
  • 25
    Song X, Zeng L, Pilo CM, Zagorski J, Wahl SM. Inhibition of bacterial cell wall-induced leukocyte recruitment and hepatic granuloma formation by TGFβ gene transfer. J Immunol 1999; 163: 40206.
  • 26
    Funk JL, Moser AH, Strewler GJ, Feingold KR, Grunfeld C. Parathyroid hormone-related protein is induced during lethal endotoxemia and contributes to endotoxin-induced mortality in rodents. Mol Med 1996; 2: 20410.
  • 27
    Funk JL, Shigenaga JK, Moser AH, Krul EJ, Strewler GJ, Feingold KR, et al. Cytokine regulation of parathyroid hormone-related protein messenger ribonucleic acid levels in mouse spleen: paradoxical effects of interferon-γ and interleukin-4. Endocrinology 1994; 135: 3518.
  • 28
    Funk JL, Trout CR, Wei H, Stafford G, Reichlin S. Parathyroid hormone-related protein (PTHrP) induction in reactive astrocytes following brain injury: a possible mediator of CNS inflammation. Brain Res 2001; 915: 195209.
  • 29
    Paul-Clark MJ, Mancini L, DelSoldata P, Flower RJ, Perretti M. Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis. Proc Natl Acad Sci U S A 2002; 99: 167782.
  • 30
    Tordjman C, Lhumeau A, Pastoureau P, Meunier F, Serkiz B, Volland JP, et al. Evaluation and comparison of urinary pyridinium crosslinks in two rat models of bone loss—ovariectomy and adjuvant polyarthritis—using a new automated HPLC method. Bone Miner 1994; 26: 15567.
  • 31
    Okazaki Y, Tsurukami H, Nishida S, Okimoto N, Aota S, Takeda S, et al. Prednisolone prevents decreases in trabecular bone mass and strength by reducing bone resorption and bone formation defect in adjuvant-induced arthritic rats. Bone 1998; 23: 35360.
  • 32
    Zernicke RF, Wohl GR, Greenwald RA, Moak SA, Leng W, Golub LM. Administration of systemic matrix metalloproteinase inhibitors maintains bone mechanical integrity in adjuvant arthritis. J Rheumatol 1997; 24: 132431.
  • 33
    Schlayer HJ, Laaff H, Peters T, Woort-Menker M, Estler HC, Karck U, et al. Involvement of tumor necrosis factor in endotoxin-triggered neutrophil adherence to sinusoidal endothelial cells of mouse liver and its modulation in acute phase. J Hepatol 1988; 7: 23949.
  • 34
    Fisker S, Kudahl K, Sonne O. Isolation of rat peritoneal mononuclear and polymorphonuclear leucocytes on discontinuous gradients of Nycodenz. J Immunol Methods 1990; 133: 318.
  • 35
    Harvath L, Falk W, Leonard EJ. Rapid quantitation of neutrophil chemotaxis: use of a polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly. J Immunol Methods 1980; 37: 3945.
  • 36
    Sugawara T, Miyamoto M, Takayama S, Kato M. Separation of neutrophils from blood in human and laboratory animals and comparison of the chemotaxis. J Pharmacol Toxicol Methods 1995; 33: 91100.
  • 37
    Williams RO, Feldmann M, Maini RN. Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis. Proc Natl Acad Sci U S A 1992; 89: 97848.
  • 38
    Joosten LAB, Helsen MM, van de Loo FAJ, van den Berg WB. Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti–tumor necrosis factor α, anti–interleukin-1α/β, and interleukin-1 receptor antagonist. Arthritis Rheum 1996; 39: 797809.
  • 39
    Clark RL, Cuttino JT Jr, Anderle SK, Cromartie WJ, Schwab JH. Radiologic analysis of arthritis in rats after systemic injection of streptococcal cell wall. Arthritis Rheum 1979; 22: 2535.
  • 40
    Takeicho O, Saito I, Tsurumachi T, Moro I, Saito T. Expression of inflammatory cytokine genes in vivo by human alveolar bone-derived polymorphonuclear leukocytes isolated from chronically inflamed sites of bone resorption. Calcif Tissue Int 1996; 58: 2448.
  • 41
    Pillinger MH, Abramson SB. The neutrophil in rheumatoid arthritis. Rheum Dis Clin North Am 1995; 21: 691714.
  • 42
    Guise TA, Yin JJ, Taylor SD, Kumagai Y, Dallas M, Boyce BF, et al. Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest 1996; 98: 15449.
  • 43
    Kinne RW, Becker W, Koscheck T, Kuhlmann J, Sharkey RM, Behr T, et al. Rat adjuvant arthritis: imaging with technetium-99m-anti-CD4 Fab′ fragments. J Nucl Med 1995; 36: 226875.
  • 44
    Van den Berg WB. Anti-cytokine therapy in chronic destructive arthritis. Arthritis Res 2001; 3: 1826.
  • 45
    Uy HL, Mundy GR, Boyce BF, Story BM, Dunstan CR, Yin JJ, et al. Tumor necrosis factor enhances parathyroid hormone-related protein-induced hypercalcemia and bone resorption without inhibiting bone formation in vivo. Cancer Res 1997; 57: 31949.
  • 46
    Sato K, Fujii Y, Kasono K, Ozawa M, Imamura H, Kanaji Y, et al. Parathyroid hormone-related protein and interleukin-1α synergistically stimulate bone resorption in vitro and increase the serum calcium concentration in mice in vivo. Endocrinology 1989; 124: 21728.
  • 47
    Zeimer HJ, Greenaway TM, Slavin J, Hards DK, Zhou H, Doery JC, et al. Parathyroid hormone-related protein in sarcoidosis. Am J Pathol 1998; 152: 1721.
  • 48
    Fradkin JM, Braniecki AM, Craig TM, Ramiro-Ibanez F, Rogers KS, Zoran DL. Elevated parathyroid hormone-related protein and hypercalcemia in two dogs with schistosomiasis. J Am Anim Hosp Assoc 2001; 37: 34955.
  • 49
    Sellars RS, Toribio RE, Blomme EA. Idiopathic systemic granulomatous disease and macrophage expression of PTHrP in a miniature pony. J Comp Pathol 2001; 125: 2148.
  • 50
    Davis JP, Archer DJ. Fisher C, Wimalawansa SJ, Baldwin D. Multiple recurrent giant cell lesions associated with high circulating levels of parathyroid hormone-related protein in a young adult. Br J Oral Maxillofac Surg 1991; 29: 1025.
  • 51
    Hirsch E, Katanaev VL, Garlanda C, Azzolino O, Pirola L, Silengo L, et al. Central role for G protein-coupled phosphoinositide 3-kinase γ in inflammation. Science 2000; 287: 104953.
  • 52
    Ritchie CK, Thomas KG, Andrews LR, Tindall DJ, Fitzpatrick LA. Effects of the calciotrophic peptides calcitonin and parathyroid hormone on prostate cancer growth and chemotaxis. Prostate 1997; 30: 1837.
  • 53
    Halstead LR, Lai CF, Cheng SL. PTH stimulates chemotaxis of human osteoblasts vis protein kinase C pathway [abstract]. J Bone Miner Res 2001; 16 Suppl 1: S498.
  • 54
    Horowitz MC, Coleman DL, Flood PM, Kupper TS, Jilka RL. Parathyroid hormone and lipopolysaccharide induce murine osteoblast-like cells to secrete a cytokine indistinguishable from granulocyte-macrophage colony-stimulating factor. J Clin Invest 1989; 83: 14957.
  • 55
    Weir EC, Insogna KL, Horowitz MC. Osteoblast-like cells secrete granulocyte-macrophage colony-stimulating factor in response to parathyroid hormone and lipopolysaccharide. Endocrinology 1989; 124: 899904.
  • 56
    Funk JL. A role for parathyroid hormone-related protein in the pathogenesis of inflammatory/autoimmune diseases. Int Immunopharmacol 2001; 1: 110121.
  • 57
    Funk JL, Migliati E, Chen G, Wei H, Wilson J, Downey KJ, et al. Parathyroid hormone-related protein induction in focal stroke: a neuroprotective vascular peptide. Am J Physiol Regul Integr Comp Physiol 2003; 284: R102130.
  • 58
    Kukreja SC, Rosol TJ, Wimbiscus SA, Shevrin DH, Grill V, Barengolts EI, et al. Tumor resection and antibodies to parathyroid hormone-related protein cause similar changes on bone histomorphometry in hypercalcemia of cancer. Endocrinology 1990; 127: 30510.
  • 59
    Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J, Schwieterman WD, et al. Tuberculosis associated with infliximab, a tumor necrosis factor α-neutralizing agent. N Engl J Med 2001; 345: 1098104.