PATIENTS AND METHODS

Patients.

Tissue specimens were obtained from patients with RA and from patients with osteoarthritis (OA) as a control. Peripheral blood (PB) and bone marrow (BM) samples were obtained from the same patients during total hip replacement surgery. Included were 65 patients with RA (51 women and 14 men, median age 57.5, range 24-72) and 63 patients with OA (43 women and 20 men, median age 55, range 24-74. 33 RA patients were treated both with methotrexate (MTX) and steroids, 5 received only methotrexate and 17 received only steroids. The remaining patients were on nonsteroidal anti-inflammatory drugs (NSAID). Patients fulfilled the American College of Rheumatology revised criteria for RA or for OA.

In addition, bone marrow trephines of 98 patients with RA were retrieved from the files of Department of Pathology. All samples were submitted in order to establish potential hematological disease diagnosis during the year 2003- 2009 and studied in accordance with national ethical principles. Fifteen patients fulfilling the American College of Rheumatology revised criteria for RA with Larsen score 1 or 2, and associated with mild, unexplained hematological abnormalities such as anemia, leucopenia, thrombocytopenia, eosinophilia, monoclonal gammapathy or splenomegaly were selected. The patients median age 51 (range 44-74) years. Clinical data of these patients are shown in table 1.

Additionally, 6 bone marrow samples were randomly selected from 27 patients previously studied by flow cytometry for immunohistochemical evaluation.

All individuals gave informed consent and the study was approved by the Institute of Rheumatology Ethics Committee.

Table 1. Basic clinical data of 15 patients with rheumatoid arthritis (Larsen score 1 or 2)

Case / Age/
gender / Disorder/ indication for bone marrow
biopsy / Final hematological diagnosis / Radiological Larsen score / RA duration
(months)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15 / 61/F
71/F
53/F
51/F
61/F
46/F
73/F
44/M
50/F
50/F
48/M
74/F
64/F
49/F
47/F / anemia
anemia
monoclonal gammapathy
leucopenia
leucopenia,
thrombocytopenia
anemia,
leucopenia
anemia,
leucopenia
leucopenia
thrombocytopenia
splenomegaly
eosinophilia
leucopenia,
thrombocytopenia
leucopenia,
monocytosis
eosinophilia
leucopenia / anemia of chronic disease
anemia of chronic disease
monoclonal gammapathy of undetermined significance
immunological leucopenia
secondary myelodysplasia
megaloblastic anemia
secondary myelodysplasia
secondary myelodysplasia
idiopathic thrombocytopenia
splenomegaly secondary to RA
reactive eosinophilia
secondary myelodysplasia
secondary myelodysplasia
reactive eosinophilia
immunological leucopenia / I
II
I
II
II
II
I
II
I
I
II
I
II
II
I / 18
24
16
21
27
14
18
37
24
19
35
12
34
28
12

F, female; M, male;

Materials and methods

Flow cytometry

Fluorescent-labeled antibodies: CD3 FITC, CD3 PE, CD25 PE, CD69 APC, CD4 PerCP, CD8 PerCP, CD4 APC, and CD8 APC were purchased from Becton Dickinson (San Jose, CA, USA). CFSE was purchased from Molecular Probes (Eugene, OR, USA) Data were acquired and analyzed using FACSCalibur and CellQuest software (Becton Dickinson). Absolute cell number was assessed using Trucount beads (Becton Dickinson) according to manufacturer instructions. IL-15Rα surface expression was analyzed on cells separated by gradient centrifugation. After acid wash of surface bound IL-15 cells were stained with anti-IL-15Ra Ab (R&D Systems, MN, USA) followed by flow cytometric analysis as described previously.[14]

IL-15 detection by ELISA

Bone marrow samples were diluted four times in PBS (Sigma, St Louis, Missouri, USA) with sodium citrate as anticoagulant. Bone marrow plasma samples were obtained by centrifugation. The bone marrow plasma concentrations of IL-15 were measured by enzyme-linked immunosorbent assay (ELISA), performend in duplicates. Polyclonal goat IgG anti-IL-15 (R&D Systems) was used as a coating Ab. Next, the biotynylated polyclonal rabbit anti-IL-15 (Peprotech, NJ, USA) was added as a detecting Ab, followed by incubation with streptavidin-peroxidase (Sigma). Recombinant human IL-15 (Genzyme, Cambridge, MA, USA) was used as a standard. The peroxidase reaction was developed using o-phenylenediamine dihydrochloride as a substrate (Sigma). The optical density was measured at 492 nm with an automatic ELISA reader (LP 400, Diagnostic Pasteur, Marnes la Coquette, France). The detection limit was 15 pg/ml.

IL-7 detection by ELISA

The bone marrow plasma concentrations of IL-7 were measured by enzyme-linked immunosorbent assay (ELISA) Quantikine test (R&D Systems) according to manufacturer’s instructions. The detection limit was 31 pg/ml.

Proliferation assays.

Bone marrow mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare Bio-Sciences, Uppsala, Sweden). Washed cells were stained with CFSE as described previously.[1] Labeled cells (2x106/ml) were cultured in 24 well plates (Nunc, Roskilde, Denmark) in RPMI 1640 media (Invitrogen, Paisley, England) supplemented with 2 mM l-glutamine (Invitrogen), 10% heat inactivated FCS (Biochrom AG, Berlin, Germany) 100U/ml penicillin, 100ug/ml streptomycin (both antibiotics from Polfa Tarchomin, Warsaw, Poland), 30µg/ml kanamycin (Sigma) and 1mM HEPES (Invitrogen) for 72 hours. After stimulation with IL-15 5 ng/ml (R&D Systems) or media (control) cells were collected and analyzed by flow cytometry.

Immunohistochemistry

Trephine biopsies obtained from iliac crest and bone marrow biopsies obtained during hip replacement surgery were histopathologically examined. They were fixed in Oxford fixative (formaldehyde 40%, glacial acetic acid, sodium chloride, distilled water), routinely processed and embedded in paraffin wax. Sections 2 mm thick were cut and stained with hematoxylin and eosin. In all cases immunohistochemical studies were done (EnVisionTM Detection Systems) (Dako Denmark A/S, Glostrup, Denmark) (DAKO) using the following mono- and polyclonal antibodies: CD3 (polyclonal), CD20 (L26), CD8 (C8/144B) (DAKO) and CD4 (4B12), CD69 (CH11 ) (Novocastra, now part of Leica Microsystems, Wetzlar, Germany) as well as anti-human interleukin -15 (IL-15, polyclonal) (R&D Systems, MN, USA). Positive and negative controls were included. The source and specificity of each antibody reagent as well as internal positive controls are shown in table 2 and 3. Degree and pattern of T-cell (CD3+) and B-cell (CD20+) infiltration in bone marrow were identified. The T-cell population was further defined using antibodies to helper/inducer and suppressor/cytotoxic T lymphocyte- associated membrane antigens, respectively CD4 and CD8. Antibody against CD69 was used to highlight recently activated T and B lymphocytes. Moreover expression of IL-15 on lymphoid cells was assessed.

Table 2. Monoclonal and polyclonal antibody reagents used in the present study

Antibody / Clone / Symbol / Dilution / Source / Reactivity
CD20 / L 26 / M0755 / 1:50 / DAKO / Precursors and mature B cells
CD3 / polyclonal / A0452 / 1:50 / DAKO / Pan T-cell marker
CD8 / C8/144B / M7103 / 1:20 / DAKO / Mature suppressor/cytotoxic T- cells, subgroup of NK cells, majority of thymocytes
CD4 / 4B12 / NCL-L-CD4-368 / 1:10 / Novocastra / Helper/inducer T- cells, monocytes
CD69 / CH11 / NCL-CD69 / 1:50 / Novocastra / Activated T - and B-cells
Human IL-15 / polyclonal / AF315 / 1:10 / R&D Systems / Cells and tissues expressing IL-15

Table 3. The tissues used as positive controls.

Antibody / Type of tissue / Internal positive control
CD20 / human tonsil / B zone cells
CD3 / human tonsil / T zone cells
CD8 / human tonsil / T zone cells
CD4 / human tonsil / T zone cells
CD69 / human tonsil / activated T and B cells
IL-15 / rheumatoid arthritis synovial membrane / synovial cells

REFERENCES

1. Rudnicka W, Burakowski T, Warnawin E et al. Functional TLR9 modulates bone marrow B cells from rheumatoid arthritis patients. Eur J Immunol 2009;39:1211-20.

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