Synopsis
Introduction
Aims
Methods
Results
Percent of positive cells | Mean fluorescence intensity | |||
---|---|---|---|---|
Composition | Mean ± SEM | Range | Mean ± SEM | Range |
(a) Primary-culture RA synovial cells | ||||
Thy-1 (CD90) (n = 4) | 62.0 ± 14.6 | 24–93 | 152 ± 49 | 46–283 |
CD14 (n = 4) | 14.8 ± 6.0 | 2–25 | 230 ± 87 | 64–445 |
(b)RA-SFB (negative isolation from primary culture; negative fraction) | ||||
Thy-1 (n = 9) | 73.8 ± 9.6 | 21–99 | 109 ± 20 | 53–222 |
Prolyl-4-hydroxylase (n = 9) | 84.8 ± 2.9 | 66–95 | 164 ± 45 | 20–391 |
CD14 (n = 9) | 0.7 ± 0.2 | 0.1–1.7 | 62 ± 12 | 30–126 |
CD68 (mAb PG-M1) (n = 5) | 1.7 ± 0.6 | 0.4–3.2 | 120 ± 78 | 10–430 |
(c)RA-SFB (fourth-passage; isolated cells) | ||||
Thy-1 (n = 3) | 86.6 ± 4.6 | 79–95 | 41 ± 7 | 27–53 |
Prolyl-4-hydroxylase (n = 3) | 98.7 ± 0.8 | 97–100 | 164 ± 62 | 82–285 |
CD14 (n = 3) | 0.6 ± 0.2 | 0.2–1.0 | 50 ± 30 | 20–110 |
(d)RA-SFB (fourth-passage; conventionally passaged cells) | ||||
Thy-1 (n = 6) | 85.9 ± 3.9 | 70–95 | 95 ± 15 | 43–167 |
Prolyl-4-hydroxylase (n = 6) | 98.3 ± 0.6 | 95–99 | 189 ± 75 | 20–518 |
CD14 (n = 6) | 1.6 ± 0.6 | 0.2–4.9 | 83 ± 32 | 22–255 |
CD68 (mAb PG-M1) (n = 3) | 0.5 ± 0.0 | 0.5–0.6 | 29 ± 6 | 19–40 |
(e)OA-SFB (negative isolation from primary culture; negative fraction) | ||||
Thy-1 (n = 9) | 62.1 ± 9.0 | 28–92 | 120 ± 36 | 53–404 |
Prolyl-4-hydroxylase (n = 7) | 92.2 ± 2.8 | 77–98 | 212 ± 44 | 30–382 |
CD14 (n = 9) | 1.3 ± 0.3 | 0.7–3.0 | 268 ± 97 | 14–754 |
CD68 (mAb PG-M1) (n = 7) | 1.3 ± 0.2 | 0.6–2.1 | 58 ± 18 | 14–140 |
(f)OA-SFB (fourth-passage; conventionally passaged cells) | ||||
Thy-1 (n = 3) | 80.9 ± 5.0 | 72–93 | 56 ± 6 | 46–70 |
Prolyl-4-hydroxylase (n = 3) | 98.8 ± 0.3 | 98–99 | 250 ± 27 | 205–313 |
CD14 (n = 3) | 1.0 ± 0.5 | 0.2–2.2 | 24 ± 1 | 22–26 |
(g) Primary culture or first-passage normal skin-FB | ||||
Thy-1 (n = 8) | 91.4 ± 3.0 | 78–99 | 156 ± 36 | 34–349 |
Prolyl-4-hydroxylase (n = 8) | 70.7 ± 15.2 | 2–99 | 101 ± 31 | 10–269 |
CD14 (n = 8) | 1.5 ± 0.4 | 0.3–4.0 | 70 ± 14 | 22–130 |
CD68 (mAb PG-M1) (n = 5) | 0.5 ± 0.3 | 0.1–1.4 | 82 ± 25 | 12–164 |
Discussion
Introduction
Materials and methods
Patients
Disease | ||||||||
---|---|---|---|---|---|---|---|---|
Patients | Gender | Age | duration | RF | ESR | CRP | Number of ARA | Concomitant |
(total) | (m/f) | (years) | (years) | (+/-) | (mm/h) | (mg/l) | criteria (RA) | medication |
Rheumatoid arthritis | ||||||||
16 | 7/9 | 61.4 ± 2.2 | 12.0 ± 2.4 | 11/4 (n.d. = 1) | 33.0 ± 4.6 | 41.8 ± 7.7 | 5.2 ± 0.2 | MTX (n = 10) |
Prednisolone (n = 13) | ||||||||
Sulfasalazine (n = 4) | ||||||||
Gold salts (n = 1) | ||||||||
NSAIDs (n = 12) | ||||||||
Osteoarthritis | ||||||||
21 | 5/16 | 70.4 ± 2.6 | 9.8 ± 3.0 | 0/8 (n.d. = 13) | 15.6 ± 1.9 | 4.6 ± 1.4 | 0.3 ± 0.1 | MTX (n = 1) |
Prednisolone (n = 1) | ||||||||
NSAIDs (n = 11) | ||||||||
None (n = 10) |
Histochemical detection of non-specific esterase
Immunohistochemistry
Antibody (technique) | Antigen recognized | Cellular localization | Main cellular expression |
---|---|---|---|
MOPC 21 (F/I) | - | [IgG1 isotype control] | |
UPC-10 (F/I) | - | [IgG2a isotype control] | |
Rabbit Ig/rabbit serum (F/I) | - | [rabbit Ig control] | |
Monoclonal | |||
AS02 (F/I) | Thy-1 (CD 90) | Membrane | Fibroblasts, endothelial cells, thymocytes |
W6/32 (F) | MHC-I (A, B, C) | Membrane | Ubiquitous |
CII 1.47 (F) | MHC-II (Pan) | Membrane | Antigen presenting cells, fibroblasts |
SJD1 (F) | CD13 | Membrane | Macrophages, fibroblasts, PMN, T cells |
3-2B12 (F/I) | Prolyl-4-hydroxylase | Cytoplasm | Fibroblasts, endothelial cells |
V9 (F) | Vimentin | Cytoplasm | Fibroblasts, endothelial cells |
4F9 (F/I) | von Willebrand factor | Cytoplasm | Endothelial cells |
UCHT-1 (F) | CD3 | Membrane | T cells |
TM316 (F) | CD11b | Membrane | Monocytes/macrophages, PMN, NK cells |
MoS39 (F/I) | CD14 | Membrane | Monocytes/macrophages |
Tyk 4 (F/I) | CD14 | Membrane | Monocytes/macrophages |
80H5 (F) | CD15 | Membrane | PMN, monocytes |
HD 37 (F) | CD19 | Membrane | B cells |
B-Ly 1 (F) | CD20 | Membrane | B cells |
Cadherin 5(F/I) | CD144 | Membrane | Endothelial cells |
AT 13/5 (F) | CD38 | Membrane | Plasma cells, activated T cells |
NKH/1 (F) | CD56 | Membrane | NK cells |
PG-M1 (F/I) | CD68 | Membrane/cytoplasm | Macrophages* |
HB 15a (F) | CD83 | Membrane | Dendritic cells, activated lymphocytes |
BBIG-V1(F/I) | VCAM-1 (CD 106) | Membrane | Fibroblasts, endothelial cells |
1.4C3 (F) | VCAM-1 (CD 106) | Membrane | Fibroblasts, endothelial cells |
Polyclonal | |||
MP I (F/I) | Procollagen I | Cytoplasm | Fibroblasts |
MP III (F/I) | Procollagen III | Cytoplasm | Fibroblasts |
Anti-c-Fos(F) | c-Fos | Nucleus/cytoplasm | Ubiquitous |
Anti-c-Jun(F) | c-Jun | Nucleus/cytoplasm | Ubiquitous |
Tissue digestion and cell culture
Negative isolation from primary culture
Conventional isolation of RA-SFB and OA-SFB by repeated passage
Flow cytometry
Proliferation assays
Statistical analysis
Results
Expression of Thy-1 in RA synovial tissue
Phenotype analysis of RA synovial cells in primary culture
Flow cytometry/histochemical analysis of cells negatively isolated from primary culture
In vitro morphology of negatively isolated RA-SFB upon reculture
Phenotype characterization of negatively isolated RA-SFB
skin-FB (n = 8) | OA-SFB (n = 9) | RA-SFB (n = 9) | ||||
---|---|---|---|---|---|---|
Antigen | Percent | MFI | Percent | MFI | Percent | MFI |
Thy-1 (CD90; mAb AS02) | 91.4 ± 3.0 | 156 ± 36 | 62.1 ± 9.0# | 120 ± 36 | 73.8 ± 9.6 | 109 ± 20 |
MHC-I | 90.2 ± 8.5 | 414 ± 119 | c87.7 ± 6.8 | 759 ± 170 | a77.2 ± 8.1 | 130 ± 54x |
MHC-II | 1.5 ± 0.4 | 225 ± 67 | c17.0 ± 7.3# | 678 ± 137# | a65.8 ± 12.3#x | 223 ± 84x |
Aminopeptidase N (CD13) | b84.0 ± 10.2 | 365 ± 163 | a83.2 ± 8.7 | 794 ± 207 | 84.8 ± 5.9 | 444 ± 226 |
VCAM-1 (CD 106) | c8.3 ± 3.4 | 66 ± 33 | a28.9 ± 13.2 | 156 ± 82 | c17.5 ± 9.8 | 122 ± 42 |
Prolyl-4-hydroxylase | 70.7 ± 15.2 | 101 ± 31 | a92.2 ± 2.8 | 212 ± 44 | 84.8 ± 2.9 | 164 ± 45 |
Vimentin | a90.3 ± 4.2 | 133 ± 34 | b94.2 ± 1.7 | 1083 ± 530 | a93.2 ± 1.5 | 592 ± 290 |
Procollagen I | d68.2 ± 8.3 | 18 ± 1 | d27.5 ± 10.4 | 39 ± 5# | a44.8 ± 13.0 | 25 ± 2x |
Procollagen III | d55.1 ± 9.8 | 15 ± 2 | d22.1 ± 7.8 | 45 ± 9# | a50.0 ± 14.3 | 26 ± 3# |
c-Fos | d87.2 ± 2.1 | 17 ± 3 | d53.9 ± 10.4 | 35 ± 2# | a57.4 ± 14.0 | 20 ± 2x |
c-Jun | d42.7 ± 15.6 | 13 ± 3 | d22.2 ± 10.3 | 29 ± 3 | b42.5 ± 14.0 | 21 ± 5 |
Jun-D | d87.4 ± 1.5 | 19 ± 5 | d37.6 ± 17.7 | 33 ± 1# | a53.0 ± 14.8 | 21 ± 4 |
Surface antigens
Cytoplasmic antigens
Cytoplasmic/intranuclear antigens
Comparison of FACS analysis and immunohistochemistry
Phenotype comparison of primary-culture and fourth-passage RA-SFB
Isolated primary RA-SFB versus conventional fourth-passage RA-SFB
Primary-culture | Fourth-passage | Fourth-passage | ||||
---|---|---|---|---|---|---|
RA-SFB | RA-SFB | RA-SFB | ||||
Isolated cells | Isolated cells | Conventionally passaged cells | ||||
(n = 9 patients) | (n = 3 patients) | (n = 6 patients) | ||||
Antigen | Percent | MFI | Percent | MFI | Percent | MFI |
Thy-1 (CD90; mAb AS02) | 73.8 ± 9.6 | 109 ± 20 | 86.6 ± 4.6 |
41 ± 7
#
| 85.9 ± 4.3 |
95 ± 17
x
|
MHC-I | a77.2 ± 8.1 | 130 ± 54 |
99.2 ± 0.2
#
| 117 ± 48 |
97.4 ± 1.4
#
| 175 ± 78 |
MHC-II | a65.8 ± 12.3 | 223 ± 84 |
4.7 ± 2.9
#
| 59 ± 25 |
4.8 ± 2.4
#
| 80 ± 21 |
Aminopeptidase N (CD13) | 84.8 ± 5.9 | 444 ± 226 | 97.2 ± 1.6 | 108 ± 49 |
97.9 ± 1.2
#
| 236 ± 95 |
VCAM-1 (CD106) | c17.5 ± 9.8 | 122 ± 42 | n.d. | n.d. | c0.6 ± 0.1 |
21 ± 5
#
|
Prolyl-4-hydroxylase | 84.8 ± 2.9 | 164 ± 45 |
98.7 ± 0.8
#
| 164 ± 62 |
98.3 ± 0.7
#
| 189 ± 82 |
Vimentin | a93.2 ± 1.5 | 592 ± 290 |
99.5 ± 0.4
#
| 717 ± 126 |
99.7
#
| 325 |
Procollagen I | a44.8 ± 13.0 | 25 ± 2 |
92.7 ± 2.8
#
| 25 ± 12 |
91.0 ± 4.0
#
| 28 ± 5 |
Procollagen III | a50.0 ± 14.3 | 26 ± 3 |
94.1 ± 1.8
#
| 21 ± 12 |
90.2 ± 5.2
#
| 22 ± 4 |
c-Fos | a57.4 ± 14.0 | 20 ± 2 | 63.5 ± 16.4 | 10 ± 5 |
95.0 ± 0.6
#x
| 17 ± 2 |
c-Jun | b42.5 ± 14.0 | 21 ± 5 | n.d. | n.d. | d42.1 ± 12.0 |
9 ± 2
#
|
Jun-D | a53.0 ± 14.8 | 21 ± 4 | 68.1 ± 13.2 | 11 ± 5 |
93.0 ± 1.3
#x
| 18 ± 3 |