PBMCs from GPA patients form large TRAP+ MNGs and degrade bone matrix.

<p>PBMCs from both GPA patients and healthy controls were cultured in the presence of 25 ng/ml M-CSF and 100 ng/ml RANKL, and stained for the presence of TRAP expression after 9 days in culture. Nuclei were counterstained with hematoxylin. MNGs with three or more nuclei and TRAP expression (purple cytoplasmic stain) were counted. Few MNGs were generated at day 9 from the PBMCs of healthy controls (A); in contrast, large MNG with numerous nuclei were formed from patients with GPA (representative field shown in B). Further, the generated cells were able to degrade bone matrix. Numerous large pits were formed by GPA PBMCs (D) as compared to the healthy control PBMCs after 9 days (C). C and D are representative of 3 independent experiments. Original magnification x 10.</p

Lack of correlation is noted between MNG formation and circulating monocyte frequency in GPA.

<p>PBMC of healthy controls (N = 10), localized (N = 5) and systemic GPA (N = 8) were stained with anti-human CD14 antibodies and the percentage of CD14+ monocytes were determined using FACS. The number of the circulating monocytes did not differ between three groups (A). Further, percentage of CD14+ monocytes were plotted against formed MNGs per 2×10⁵ PBMC. There was no correlation between percentage of CD14+ monocytes in PBMCs and formed MNGs in both localized and systemic GPA (r² = 0.19, P = 0.46 for localized GPA; r² = 0.17, P = 0.30 for systemic GPA), indicating that systemic GPA formed higher number of MNGs per equivalent number of circulating CD14+ monocytes (B).</p

MNGs express cathepsin K.

<p>MNGs were evaluated for the expression of phenotypic markers of osteoclast cathepsin K and calcitonin-receptor using immunohistochemistry. The slides were counter-stained with hematoxylin. MNGs in the GPA granulomata expressed cathepsin K (A, brown cytoplasmic stain). However, it was not universally noted in all MNGs (A, arrow). MNGs (*) did not express calcitonin-receptors (B). Original magnification x 10.</p

MNGs in the lung of patients with GPA express TRAP.

<p>Alveolar macrophages in control (A) and unaffected tissue in GPA lung (B) expressed TRAP (bright purple), and MNGs were not noted in normal lung tissue. A population of MNGs within the GPA granulomata expressed TRAP. TRAP expressing MNGs were localized in close vicinity to geographic necrosis (C) while MNGs were rarely seen in regions with few inflammatory infiltrates (D). MNGs varied in size and in number of incorporated nuclei. Further, the expression of TRAP varied between MNGs (E and F). Original magnification X 5 for C and D; X 10 for A, B and E; X 20 for F, respectively.*, TRAP positive macrophages; arrows, MNG; arrow heads, geographic necrosis.</p

Increased MNG formation in patients with systemic GPA compared to those with limited disease.

<p>PBMC of healthy controls (N = 11) and GPA patients (N = 13) were cultured in presence of M-CSF and RANKL for 9 days and MNGs counted as in Materials and Methods. PBMCs from GPA patients generated significantly more TRAP+ MNGs than healthy controls (P = 0.022). When the GPA group was analyzed based on disease phenotype [ENT-localized (N = 5) vs. systemic form (N = 8)], only patients with systemic disease formed significantly more MNGs (P = 0.0015); no difference was seen between localized GPA and health controls (P = 0.955).</p

Pulmonary Outcomes According to PAD3/4 Cross-Reactive Antibody Status.

<p>Values are median (interquartile range) unless otherwise noted.</p><p>*p-values are for the comparison of the no anti-PAD vs. anti-PAD3/4XR groups.</p><p>PAD = peptidyl-arginine deiminase; anti-PAD3/4XR = anti-PAD3/4 Cross-Reactive antibodies; RA = rheumatoid arthritis; ILD = interstitial lung disease; GGO = ground glass opacification; R/TB/HC = reticulation/traction bronchiectasis/honeycombing; PFT = pulmonary function testing; Impaired Diff = impaired diffusion.</p

Crude and Adjusted Associations of PAD3/4 Cross-Reactive Antibodies with Pulmonary Outcomes in RA.

<p>*Adjusted for age, RA duration, RF, CCP2, IL-6, SHS.</p><p>**Adjusted for age, gender, RF, CCP2, RA duration, DAS28, prednisone use, biologic DMARD use, current and past smoking, and SHS.</p>†<p>β coefficients are Odds Ratios.</p><p>ILD = interstitial lung disease; GGO = ground glass opacification; R/TB/HC = reticulation/traction bronchiectasis/honeycombing; PFT = pulmonary function testing; Impaired Diff = impaired diffusion; RA = rheumatoid arthritis; RF = rheumatoid factor; CCP2 = 2^nd generation anti-cyclic citrullinated peptide antibody; DAS = disease activity score; CRP = C-reactive protein; IL = interleukin; SHS = Total modified Sharp-van der Heijde Score; DMARD = disease modifying anti-rheumatic drug.</p

Patient Characteristics According to the Presence of CT-ILD Features.

<p>Values are mean ± standard deviation or median (interquartile range) unless otherwise noted.</p><p>RA = rheumatoid arthritis; RF = rheumatoid factor; CCP2 = 2^nd generation anti-cyclic citrullinated peptide antibody; DAS = disease activity score; CRP = C-reactive protein; IL = interleukin; SHS = Total modified Sharp-van der Heijde Score; HAQ = Health Assessment Questionnaire Disability Index; TNF = tumor necrosis factor; DMARD = disease modifying anti-rheumatic drug; ILD = interstitial lung disease; PFT = pulmonary function testing; Impaired Diff = impaired diffusion.</p

Characteristics of GPA patients.

<p>Yrs, years; F, female; M, male; BVAS-WG, Birmingham Vasculitis Activity Score-Wegener’s Granulomatosis; TRAP, tartrate-resistant acid phosphatase; MNG, multi-nucleated giant cells; AZA, Azathioprine; P, Prednisone; RTX, Rituximab, CTX, cyclophosphamide; MMP, mycophenolate mofetil.</p

Patient Characteristics According to PAD3/4 Cross-Reactive Antibody Status.

<p>Values are mean ± standard deviation or median (interquartile range) unless otherwise noted.</p><p>*p-values are for the comparison of the no anti-PAD vs. anti-PAD3/4XR groups.</p><p>PAD = peptidyl-arginine deiminase; anti-PAD3/4XR = anti-PAD3/4 Cross-Reactive antibodies; RA = rheumatoid arthritis; RF = rheumatoid factor; CCP2 = 2^nd generation anti-cyclic citrullinated peptide antibody; DAS = disease activity score; CRP = C-reactive protein; IL = interleukin; SHS = Total modified Sharp-van der Heijde Score; HAQ = Health Assessment Questionnaire Disability Index; TNF = tumor necrosis factor; DMARD = disease modifying anti-rheumatic drug.</p