Distinct vascular lesions in giant cell arteritis share identical T cell clonotypes.

Giant cell arteritis (GCA) is a spontaneous vasculitic syndrome that specifically targets the walls of medium and large arteries. Vascular lesions are characterized by patchy granulomatous infiltrates composed of T cells, macrophages, histiocytes, and giant cells. To test the hypothesis that a locally residing antigen recruits T cells into the vessel walls, we have analyzed T cell receptor (TCR) molecules of tissue infiltrating T cells. A total of 638 CD4+ T cell clones were isolated from temporal artery specimens of three patients with GCA. Analysis of TCR molecules for the usage of V beta 1-V beta 20 revealed that all TCR V beta elements were represented, demonstrating that interleukin 2 (IL-2)-responsive T cells infiltrating the tissue are highly diverse. To detect expanded T cell specificities, we made use of the patchy character of the inflammatory disease and compared the TCR repertoire of T cells established from independent vasculitic foci of the same artery. Sequence analysis of TCR V beta chains documented that individual TCR specificities were present in multiple copies, indicating clonal expansion. T cells with identical beta chains were isolated from distinct inflammatory foci of the same patient. These specificities represented only a small fraction of tissue-infiltrating T cells and involved the V beta 5.3 gene segment in the two patients sharing the HLA-DRB1*0401 allele. The third complementarity determining region of clonally expanded TCR beta chains was characterized by a cluster of negatively and positively charged residues, suggesting that the juxtaposed antigenic peptide is charged. The sharing of identical T cell specificities by distinct and independent regions of the granulomatous inflammation suggests that these T cells are disease relevant and that their repertoire is strongly restricted. These data suggest that an antigen residing in the arterial wall is recognized by a small fraction of CD4+ T cells in the inflammatory process characteristic for GCA.

The HLA-DRB1 locus as a genetic component in giant cell arteritis. Mapping of a disease-linked sequence motif to the antigen binding site of the HLA-DR molecule.

Giant cell arteritis (GCA) is a granulomatous vasculitis affecting persons over 50 years of age. The inflammatory infiltrate, which is targeted at the aorta and its proximal branches, includes activated CD4+ helper T cells, histiocytes, and giant cells. To investigate whether the genetic polymorphism of the HLA-DRB1 genes contributes to the local accumulation of activated T cells, we have analyzed both HLA-DRB1 alleles in a cohort of 42 patients with biopsy-proven GCA. The majority of patients (60%) expressed the B1*0401 or B1*0404/8 variant of the HLA-DR4 haplotype, both of which also represent the major genetic factors underlying the disease association in RA. GCA patients negative for the disease-linked HLA-DR4 alleles were characterized by a nonrandom distribution of HLA-DRB1 alleles. Sequence comparison among the allelic products identified in the GCA cohort demonstrated heterogeneity for the sequence polymorphism of the third hypervariable region (HVR), but homology for the polymorphic residues within the HVR2 of the HLA-DRB1 gene. The GCA patients shared a sequence motif spanning amino acid positions 28-31 of the HLA-DR beta 1 chain. In the structural model for HLA-DR molecules, this sequence motif can be mapped to the antigen-binding site of the HLA complex, suggesting a crucial role of antigen selection and presentation in GCA. In contrast, the sequence polymorphism linked to RA has been mapped to the HVR3 of the HLA-DRB1 gene and translates into a distinct domain of the HLA-DR molecule, the alpha-helical loop surrounding the antigen-binding groove. A consecutive case series study demonstrated that GCA and RA rarely co-occurred, supporting the interpretation that distinct functional domains of the HLA-DR molecule are implicated in the pathomechanisms of these two autoimmune diseases.

Effect of vitamin D pretreatment of human mesenchymal stem cells on ectopic bone formation.

Adult mesenchymal stem cells (MSCs) are used in contemporary strategies for tissue engineering. The MSC is able to form bone following implantation as undifferentiated cells adherent to hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds. Previous investigators have demonstrated that human MSCs (hMSCs) can be differentiated to osteoblasts in vitro by the inclusion of vitamin D and ascorbic acid. The aim of this study was to compare the osteogenic potential of predifferentiated and undifferentiated bone marrow-derived, culture-expanded hMSCs adherent to synthetic HA/TCP (60%/40%) following subcutaneous engraftment in severe combined immunodeficiency (SCID) mice. During the final 3 days of culture, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics or media containing 25-mM calcium supplementation with vitamin D and ascorbic acid. Four weeks following implantation in SCID mice, scoring analysis of bone formation within the cubes revealed the absence of bone formation in unloaded cubes. Bone formation compared by a qualitative bone index was 7.23% for undifferentiated cells compared to 5.20% for differentiated cells. Minimal resorption was observed at this early time point. In this ectopic model, predifferentiation using a combination of vitamin D and ascorbic acid failed to increase subsequent bone formation by implanted cells. Following implantation of hMSCs adherent to an osteoconductive scaffold, host factors may contribute dominant osteoinductive signals or impose inhibitory signals to control the fate of the implanted cell. Predifferentiation strategies require confirmation in vivo.

Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma.

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34 degrees C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5 degrees C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5 degrees C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10(-8) M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10(-8) M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5 degrees C with ascorbate and beta1-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.

Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation.

Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2-6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2-6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2-stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor gamma2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.

Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology.

Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID-1, ID-2, ID-3, and ID-4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor beta (CBFbeta), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID proteins, block differentiation along alternate pathways.

Nonrandom selection of T cell specificities in anti-HLA-DR responses. Sequence motifs of the responder HLA-DR allele influence T cell recruitment.

The self-restriction of Ag-specific T cell responses is interpreted as the result of a positive selection of the individual's T cell specificities for their compatibility with self-MHC molecules. If the T cell receptor (TCR) specificities in any given individual have an affinity for syngeneic MHC molecules, it is unclear how they interact with allogeneic MHC structures. To approach this question, we analyzed 123 alloreactive HLA-DR4 Dw4 or Dw14 specific T cell clones that were generated from responder/stimulator combinations with defined disparities in the HLA-DR beta 1-chain. Sets of T cell clones were established from three different HLA-Dw4+ responders and compared for their fine specificities. The majority of HLA-DR4 Dw14 specific T cell clones co-recognized HLA-DR1 Dw1+ (33 to 36% of all T cell clones) or HLA-DRw14 Dw16+ (26 to 33%) stimulators, both of which share very similar sequences in the third hypervariable region of the HLA-DR beta 1-chain with the HLA-DR4 alleles Dw4 and Dw14. These data suggest that sequence and structural similarities in the alpha-helical portions of the HLA-DR molecule impose a strong bias on the recognition of allotargets. The second haplotype of the responder did not appear to affect the typical fingerprint of T cell recognition except for the deletion of self-reactive TCR specificities. Nonrandom usage of TCR specificities in anti-HLA-DR responses was also found for HLA-DRw11/DRw13+ and HLA-DRw11/DR7+ T cell donors who did not share any obvious polymorphic sequence stretches with the allostimulators HLA-DR4 Dw4 or Dw14. T cell clones from an HLA-DRw11/DRw13+ responder functionally resembled the TCR specificities derived from the HLA-DR4 Dw4+ donors. T cell clones derived from an HLA-DRw11/DR7+ individual were characterized by a distinct cross-reactivity pattern preferring HLA-DRw13 Dw19+ (50 to 60%) and HLA-DR3+ (43 to 57%) stimulator cells. These findings suggest that the responder HLA-DR alleles influence the structural constraints in the recognition of allo-HLA-DR molecules in closely related and in completely disparate responder/stimulator combinations.

Effects of gonadal and adrenal androgens in a novel androgen-responsive human osteoblastic cell line.

While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR-6) that expresses a mature osteoblast phenotype and a physiological number (approximately 4,000/nucleus) of androgen receptors (AR). Treatment with 5alpha-dihydrotestosterone (5alpha-DHT) inhibited the proliferation of hFOB/AR-6 cells in a dose-dependent fashion, while it had no effect on the proliferation of hFOB cells, which express low levels of AR (<200/nucleus). In hFOB/AR-6 cells, co-treatment with the specific AR antagonist, hydroxyflutamide abolished 5alpha-DHT-induced growth inhibition. Steady-state levels of transforming growth factor-beta1 (TGF-beta1) and TGF-beta-induced early gene (TIEG) mRNA decreased after treatment of hFOB/AR-6 cells with 5alpha-DHT, suggesting a role for the TGF-beta1-TIEG pathway in mediating 5alpha-DHT-induced growth inhibition of hFOB/AR-6 cells. In support of this, co-treatment of hFOB/AR-6 cells with TGF-beta1 (40 pg/ml) reversed the 5alpha-DHT-induced growth inhibition, whereas TGF-beta1 alone at this dose had no effect on hFOB/AR-6 cell proliferation. Furthermore, treatment of hFOB/AR-6 cells with 5alpha-DHT and testosterone (10(-8) M) inhibited basal and 1,25-(OH)2D3-induced alkaline phosphatase (ALP) activity and type I collagen synthesis without affecting osteocalcin production. Thus, in this fetal osteoblast cell line expressing a physiological number of AR, androgens decrease proliferation and the expression of markers associated with osteoblast differentiation. These studies suggest that the potential anabolic effect of androgens on bone may not be mediated at the level of the mature osteoblast.

Tissue cytokine patterns in patients with polymyalgia rheumatica and giant cell arteritis.

OBJECTIVE: To analyze temporal artery specimens from patients with giant cell arteritis and polymyalgia rheumatica for the presence of inflammatory cytokines and to ascertain whether a specific cytokine pattern exists for the two conditions. DESIGN: Case series of patients having temporal artery biopsy procedures. SETTING: The outpatient clinic and the research laboratories of the Division of Rheumatology, Mayo Clinic. PATIENTS: 34 patients having temporal artery biopsy procedures: 15 patients had giant cell arteritis, 9 had polymyalgia rheumatica without evidence of vasculitis, and 10 had neither polymyalgia rheumatica nor vasculitis. MEASUREMENT: Temporal artery specimens were analyzed for in vivo presence of cytokine messenger RNA (mRNA) by polymerase chain reaction with cytokine-specific primer sets. RESULTS: Vasculitic lesions in giant cell arteritis samples were characterized by in situ production of interleukin-1 beta, interleukin-6, and transforming growth factor-beta 1 mRNA (indicative of macrophage activation) and by interferon-gamma and interleukin-2 mRNA (indicative of selective T-cell activation). However, macrophage- and T-cell-derived cytokines were also detected in temporal artery biopsy specimens from patients with polymyalgia rheumatica. Tissue-infiltrating T cells in giant cell arteritis and polymyalgia rheumatica samples each had distinctive lymphokine profiles. Although interferon-gamma was found in 67% of giant cell arteritis samples, polymyalgia rheumatica samples had only interleukin-2. CONCLUSIONS: Patients with polymyalgia rheumatica have vascular involvement. Patients with polymyalgia rheumatica and giant cell arteritis share in situ production of mRNA specific for macrophage-derived cytokines. T cells recruited to vasculitic lesions in patients with giant cell arteritis predominantly produce interleukin-2 and interferon-gamma. Patients with polymyalgia rheumatica do not have interferon-gamma production, suggesting that interferon-gamma may be involved in the progression to overt arteritis.

The influence of HLA-DRB1 genes on disease severity in rheumatoid arthritis.

OBJECTIVE: To explore the role of HLA-DRB1 genes in determining disease severity in rheumatoid arthritis. DESIGN: Case series of patients with seropositive rheumatoid arthritis. SETTING: The outpatient clinic of the Division of Rheumatology, Mayo Clinic. PATIENTS: One hundred and two patients with seropositive, erosive rheumatoid arthritis and a minimum disease duration of 3 years. MEASUREMENTS: Patients were genotyped for both HLA-DRB1 alleles and were categorized according to the expression of one or two disease-linked HLA-DRB1 alleles. Identification of HLA-DRB1 alleles was done by the polymerase chain reaction and subsequent oligonucleotide hybridization. Homozygosity for allelic variants was confirmed by sequence analysis. Immunogenetically defined patient subgroups were retrospectively evaluated for joint destruction and patterns of disease manifestation, including rheumatoid organ disease. RESULTS: Of 102 patients, 98 (96%) expressed the disease-linked sequence polymorphism. Forty-seven patients (46%) carried a double dose of the relevant sequence stretch: Twenty-eight patients expressed HLA-DRB1*04 variants on both alleles, and 19 combined an HLA-DRB*04 variant with HLA-DRB1*0101 or DRB1*1402. Nodular disease was present in 100% of patients typed as HLA-DRB1*04/04 and in 59% of patients typed as HLA-DRB1*04 and who had inherited only a single dose of the disease-linked sequence polymorphism (P < 0.0001). Major organ systems were involved in 61% and 11% of these two patient groups, respectively (P < 0.0001); and joint surgery was required in 61% and 25% (P < 0.002), respectively. Patients typed as HLA-DR*04/01 had intermediate clinical courses. CONCLUSION: Genotyping patients with rheumatoid arthritis for both HLA-DRB1 alleles identifies clinical subsets with distinct profiles of disease manifestations.

Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene.

Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completeness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild-type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5 alpha-dihydrotestosterone (5 alpha-DHT) (10(-8) M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 +/- 73 (mean +/- SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 +/- 395 functional hARs/nucleus in hFOB/AR-2 cells, and 3,987 +/- 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5 alpha-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5 alpha-DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.

HLA-DRB1 alleles in polymyalgia rheumatica, giant cell arteritis, and rheumatoid arthritis.

OBJECTIVE: Immunogenetic analysis has demonstrated that giant cell arteritis (GCA) and rheumatoid arthritis (RA) are associated with 2 different domains of the HLA-DR4 molecule. The present study was undertaken to evaluate whether polymyalgia rheumatica (PMR) immunogenetically resembles GCA or RA and to determine whether expression of HLA-DRB1 alleles can be used to detect heterogeneity among PMR patients. METHODS: Forty-six patients with PMR, 52 with GCA, 122 with seropositive RA, and 72 normal individuals were genotyped for HLA-DRB1 alleles by allele-specific amplification and subsequent oligonucleotide hybridization. RESULTS: The HLA-DRB1*04 allele was the most frequent among PMR patients (67%). While the expression of allelic variants of the HLA-DR4 family was restricted to HLA-DRB1*0401 and *0404/8 in RA patients, all HLA-DRB1*04 alleles, including B1*0402 and B1*0403, were represented in the PMR group. The distribution of HLA-DRB1 alleles among HLA-DRB1*04 negative patients was similar in those with PMR and those with GCA, and could be distinguished from that in RA patients. In particular, HLA-DRB1*01 alleles, which were found in most HLA-DRB1*04 negative RA patients, were underrepresented in patients with PMR and GCA. CONCLUSION: The distribution of HLA-DRB1 alleles in PMR resembles that found in GCA. PMR and GCA share the associated sequence polymorphism encoded by the second hypervariable region (HVR) of the HLA-DRB1 gene. The HLA-DRB1 association of PMR and GCA can be distinguished from that of RA, which is linked to a sequence motif in the third HVR of DRB1 alleles. The differential role of distinct domains on HLA-DR molecules suggests that multiple biologic functions are regulated by these molecules and that they contribute differently to disease mechanisms. The similarities in the distribution of HLA-DRB1 alleles in PMR and GCA indicates that HLA-DRB1 alleles are not predictive for progression of PMR to the vasculitic lesions that are pathognomonic for GCA.