Scandinavian Journal of Rheumatology
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
The Leukocyte Protein L1 in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Osteoarthritis H. B. Berntzen, Ü. Ölmez, M. K. Fagerhol & E. Munthe To cite this article: H. B. Berntzen, Ü. Ölmez, M. K. Fagerhol & E. Munthe (1991) The Leukocyte Protein L1 in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Osteoarthritis, Scandinavian Journal of Rheumatology, 20:2, 74-82 To link to this article: http://dx.doi.org/10.3109/03009749109165280
Published online: 12 Jul 2009.
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Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [NUS National University of Singapore]
Date: 21 October 2015, At: 02:57
Scand J Rheumatol 20: 74-82, 1991
The Leukocyte Protein L1 in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Osteoarthritis H. B. BERNTZEN'.2, U. OLMEZ', M. K . FAGERHOL3, and E. MUNTHE'*
Downloaded by [NUS National University of Singapore] at 02:57 21 October 2015
'Oslo City Department of Rheumatology, Norwegian Lutheran Hospital, 'Oslo Sanitetsforening Rheumatism Hospital, 'Blood Bank and Department of Immunology, Ullevdl Hospital, Oslo.
Berntzen HB, Olmez U, Fagerhol MK, Munthe E. The Leukocyte Protein L1 in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Osteoarthritis. Scand J Rheurnatol 1991; 20: 74-82.
L1 is a major granulocyte and monocyte protein, released during activation and turnover of such cells. Blood and synovial fluid (SF) from 41 patients with rheumatoid arthritis (RA) and 6 patients with osteoarthritis (OA), were analyzed for L1 and the acute phase proteins C-reactive protein, orosomucoid, haptoglobin, a,-antitrypsin and albumin as well as for differential leukocyte count. L1 levels in plasma and SF showed highly significant differences (p < 0.0001). between the RA and O A patients. All the O A patients had normal plasma concentrations of L1 and low concentrations of L l in SF. All the RA patients had elevated plasma levels of L1 and high L1 concentrations in SF. In the RA patients, the ratios between the protein concentrations in SF and blood were 3.29 for L l and 5 0.64 for the acute phase proteins. In the SF, the L1 levels did not correlate with the rnonocyte count, while a low, positive correlation was found between L1 and the granulocyte count. The high L1 concentrations observed in SF from R A patients probably reflected an increased turnover of leukocytes in the inflamed joints. In SF from RA patients, high L1 concentrations were found in joints with a high amount of swelling. The present study suggests that L1 may represent a marker of both local and systemic inflammation. This work has been supported by a Syntex scholarship in rheumatology
Key words: The L1 protein, calprotectin, acute phase proteins, rheumatoid arthritis, osteoarthritis. synovial fluid. Hilde Berner Berntzen, Oslo Sanitetsforening Rheumatism Hospital, Akersbakken 27, N-0172 Oslo 1, Norway.
INTRODUCTION Recent studies have shown that the plasma concentration of the L1 protein may be a useful marker of inflammatory activity in patients with rheumatoid arthritis (RA) ( 1 4 ) . Compared with ESR and acute phase proteins, L1 was shown to have the highest correlations with clinical signs of joint inflammation in patients with adult and juvenile RA (3,4). Plasma levels of L1 were shown to be positively correlated with ESR, C-reactive protein (CRP), orosomucoid (i.e. a,-acid glycoprotein), haptoglobin and a,-antitrypsin. L1 is a major leukocyte protein, and it constitutes about 5% of the total cellular proteins in granulocytes (5) and about 1.6% in monocytes (Lyberg et al, unpublished observations). L1 is mainly found in the cytosol of these cells (6). The protein is also detected in mucosal squamous epithelium (7), but not in epidermis, except in several inflammatory skin diseases (8). L1 is not found in resting or activated lymphocytes (6). L1 is released from granulocytes and monocyteshacrophages during their activation (9). The protein is detected in plasma by
*Deceased
Downloaded by [NUS National University of Singapore] at 02:57 21 October 2015
S c a d J Rheumatology 20
L A in plasma and synovial fluid
use of radio immunoassay (1&12) or enzyme immunoassay (EIA) (Fagerhol et al. unpublished observations). Our group has previously found that L1 has a molecular mass of 36.5 kDa (5) and consists of a light and two heavy chains (9,13). Other investigators have reported o n new proteins called MRP-14, MRP-8, cystic fibrosis antigen (CFA), calgranulin A and B (14-17). It has been shown, however, that these proteins are identical with the L1 chains (9,18). The knowledge about the biological functions of L1 is scarce. Fungicidal and bactericidal activities have been found (19-21), however, and the term calprotectin has therefore been suggested (9,27). High concentrations of L1 have previously been found in the synovial fluid (SF) from R A patients ( 1 ) . The aim of the present study was to analyze the concentrations of L1 in plasma and SF from patients with R A and osteoarthritis (OA). Comparisons were made with the corresponding concentrations of several acute phase proteins and the differential leukocyte counts. The present study indicates that plasma L1 levels distinguish between an inflammatory and a non-inflammatory disease, and it suggests that L1 in SF may be a marker of joint inflammation.
MATERIALS A N D METHODS Pa tien ts Out- and in-patients at the Department of Rheumatology, The Norwegian Lutheran Hospital, Oslo, were studied. All the patients had swelling of a knee which required relieving synoviocentesis. Forty-one patients had definite or classic R A (22), including 30 females and 11 males, aged 19 to 87 years (median 59), with a disease duration of 3 months to 45 years (median 5 years). Twelve were seropositive for rheumatoid factor (RF), with a titer 1 64 by Waaler test, while 20 were RF positive by the Latex fixation test. Six patients, five females and one male, aged 56 to 76 years (median 64), had O A in one knee as confirmed by X-ray. The O A patients had suffered from knee symptoms for 4 to 25 years (median 8). The patients received a variety of nonsteroidal anti-inflammatory drugs and/or second line anti-rheumatic drugs. SF was obtained from knee joints at the time of synoviocentesis. The exclusion criteria were: Corticosteroid injection into the actual knee joint during the last 3 months, previous osmium acid treatment or surgical synovectomy, secondary arthrosis. suspected bacterial arthritis, knee joint trauma or ongoing systemic infection (anamnesis). Clinical examinations Before joint aspiration, all patients were examined clinically by one of the authors (H.B.B or U . O . ) . Both the amount of swelling and the degree of tenderness, was graded as none (0), slight ( l ) , moderate ( 2 ) and severe (3). Pain in the actual knee joint was estimated by a visual analogue scale. Laboratory investigations The S F was aspirated and placed in a tube with sodium ethylene diamine tetra-acetate (EDTA). Hyaluronidase (PenetraseR), 20 IEiml SF, was added, and the S F was incubated for about 0.5 h before analysis for leukocyte count and differential count. To obtain S F devoid of cells, the fluid was centrifuged at 400 x g for 10 minutes. The supernatant was analyzed for L1, CRP, orosomucoid, haptoglobin, a,-antitrypsin and albumin. S F was frozen at -70°C until analysis of L1.
75
76 H . B . Berntzen et al.
...::
10O.LlOO
-y
... ." Downloaded by [NUS National University of Singapore] at 02:57 21 October 2015
10.000
Scand J Rheumatology 20
...
...
I .
."
I00
RA PLASM
OA
RA
OA
SYNOVIAL FLUID
Fig. 1. The concentrations of L1 in plasma and synovial fluid from rheumatoid arthritis (RA) and osteoarthritis (OA) patients.
The blood samples were analyzed for L1, CRP, orosomucoid, haptoglobin, a,-antitrypsin. albumin and leukocyte counts, as well as differential counting of leukocytes. EDTA-plasma was frozen at - 70°C until analysis of L1. LL in plasma and in SF was analyzed by EIA (Fagerhol e t al, unpublished observations). In brief, wells were coated with rabbit anti-Ll IgG, and L1 in plasma or S F was detected by polyclonal rat ant-L1 and thereafter alkaline phosphatase-conjugated rabbit anti-rat IgG. The coefficient of variation is about 5% within assay and 13% between assays. Normal plasma reference values of L1 (median k 2SD) are 8&880 wg/L (median 440) for females and 15C910 pg/L (median 530) for males. C R P in serum was analyzed by turbidimetry, orosomucoid, haptoglobin and a,-antitrypsin in serum by the use of Behring (Germany) automated nephelometer, and albumin in serum by a colorimetric method using the BioRad (USA) Coomassie Blue reagent. Total leukocyte counts in blood and SF were analyzed in an electronic cell analyzer, and differential counting was performed on May-Griinwald-Giemsa stained smears. Statistics The Mann-Whitney test was used to test for differences between two groups, while KruskalWallis test was used to analyze for differences between multiple groups. The Spearman rank correlation (r,) was used in the correlation analysis, and significance of the correlations was tested by use of Student's t-test. All p values reported were two-tailed. P values < 0.05 were considered significant.
RESULTS Laboratory results L1 levels in plasma and SF showed highly significant differences (p < 0.0001) between the R A and O A patients (Table I and Fig.1). Significant differences (0.001 < p < 0.05) between the two patient groups were also found for the concentrations of orosomucoid, CRP,
L l in plasma and svnovial fluid
Scand J Rheumatology 20
Downloaded by [NUS National University of Singapore] at 02:57 21 October 2015
Table I. The concentrations of LI and acute phase proteins in blood ( B ) and synovial fluid (SF) f r o m rheumatoid arthritis ( R A ) and osteoarthritis (OA)patients (median values with the ranges in parenthesis). Reference values are given f o r the blood analysis. Significant differences between the concentrations in the R A and OA patients are indicated. Laboratory variables
RA patients (n=41)
B-L I I SF-L I I B-alb' SF-alb' B-Oroso' SF-Oroso' B-a I -at4 SF-u,-at' B-CRP' SF-CRP' B-Hapto" SF-Hapto"
9400 ( 98546078) 18156 (1951-375368) 34 (2542) 22 (l(k34) 1.62(0,863.10) 0.99 (0.462.30) 2.1 (1.2-3.7) 1.2 (0.62.5) 33 (0-117) 13 (0-71) 4.I (0.4-7.0) 1.2 (0.1-4.2)
I
kg/L. albumin - g/L. ' Orosomucoid
-
Reference values
OA patients (n=6)
630 (495- 832)"' 895 (29C-2014)"' 40 (3542)' 23 (Zk27) 0.88 (0.661.01 )" 0.49 (0.11-0.68)" 1.5 (1.1-1.5)' 0.8(0.2-1.0)* 2 ((b15)' 2 ((M)" 2.2 (1 x 3 . 3 ) ' 0.3(0.1-1.1)'
g/L. a,-antitrypsin - g/L, ' mg/L,
9"
10
35-50 0.5-1.4 0.8-1.7
< 10 0.3-1.8
' Haptoglobin - g/L.
p
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
The Leukocyte Protein L1 in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Osteoarthritis H. B. Berntzen, Ü. Ölmez, M. K. Fagerhol & E. Munthe To cite this article: H. B. Berntzen, Ü. Ölmez, M. K. Fagerhol & E. Munthe (1991) The Leukocyte Protein L1 in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Osteoarthritis, Scandinavian Journal of Rheumatology, 20:2, 74-82 To link to this article: http://dx.doi.org/10.3109/03009749109165280
Published online: 12 Jul 2009.
Submit your article to this journal
Article views: 18
View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [NUS National University of Singapore]
Date: 21 October 2015, At: 02:57
Scand J Rheumatol 20: 74-82, 1991
The Leukocyte Protein L1 in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Osteoarthritis H. B. BERNTZEN'.2, U. OLMEZ', M. K . FAGERHOL3, and E. MUNTHE'*
Downloaded by [NUS National University of Singapore] at 02:57 21 October 2015
'Oslo City Department of Rheumatology, Norwegian Lutheran Hospital, 'Oslo Sanitetsforening Rheumatism Hospital, 'Blood Bank and Department of Immunology, Ullevdl Hospital, Oslo.
Berntzen HB, Olmez U, Fagerhol MK, Munthe E. The Leukocyte Protein L1 in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Osteoarthritis. Scand J Rheurnatol 1991; 20: 74-82.
L1 is a major granulocyte and monocyte protein, released during activation and turnover of such cells. Blood and synovial fluid (SF) from 41 patients with rheumatoid arthritis (RA) and 6 patients with osteoarthritis (OA), were analyzed for L1 and the acute phase proteins C-reactive protein, orosomucoid, haptoglobin, a,-antitrypsin and albumin as well as for differential leukocyte count. L1 levels in plasma and SF showed highly significant differences (p < 0.0001). between the RA and O A patients. All the O A patients had normal plasma concentrations of L1 and low concentrations of L l in SF. All the RA patients had elevated plasma levels of L1 and high L1 concentrations in SF. In the RA patients, the ratios between the protein concentrations in SF and blood were 3.29 for L l and 5 0.64 for the acute phase proteins. In the SF, the L1 levels did not correlate with the rnonocyte count, while a low, positive correlation was found between L1 and the granulocyte count. The high L1 concentrations observed in SF from R A patients probably reflected an increased turnover of leukocytes in the inflamed joints. In SF from RA patients, high L1 concentrations were found in joints with a high amount of swelling. The present study suggests that L1 may represent a marker of both local and systemic inflammation. This work has been supported by a Syntex scholarship in rheumatology
Key words: The L1 protein, calprotectin, acute phase proteins, rheumatoid arthritis, osteoarthritis. synovial fluid. Hilde Berner Berntzen, Oslo Sanitetsforening Rheumatism Hospital, Akersbakken 27, N-0172 Oslo 1, Norway.
INTRODUCTION Recent studies have shown that the plasma concentration of the L1 protein may be a useful marker of inflammatory activity in patients with rheumatoid arthritis (RA) ( 1 4 ) . Compared with ESR and acute phase proteins, L1 was shown to have the highest correlations with clinical signs of joint inflammation in patients with adult and juvenile RA (3,4). Plasma levels of L1 were shown to be positively correlated with ESR, C-reactive protein (CRP), orosomucoid (i.e. a,-acid glycoprotein), haptoglobin and a,-antitrypsin. L1 is a major leukocyte protein, and it constitutes about 5% of the total cellular proteins in granulocytes (5) and about 1.6% in monocytes (Lyberg et al, unpublished observations). L1 is mainly found in the cytosol of these cells (6). The protein is also detected in mucosal squamous epithelium (7), but not in epidermis, except in several inflammatory skin diseases (8). L1 is not found in resting or activated lymphocytes (6). L1 is released from granulocytes and monocyteshacrophages during their activation (9). The protein is detected in plasma by
*Deceased
Downloaded by [NUS National University of Singapore] at 02:57 21 October 2015
S c a d J Rheumatology 20
L A in plasma and synovial fluid
use of radio immunoassay (1&12) or enzyme immunoassay (EIA) (Fagerhol et al. unpublished observations). Our group has previously found that L1 has a molecular mass of 36.5 kDa (5) and consists of a light and two heavy chains (9,13). Other investigators have reported o n new proteins called MRP-14, MRP-8, cystic fibrosis antigen (CFA), calgranulin A and B (14-17). It has been shown, however, that these proteins are identical with the L1 chains (9,18). The knowledge about the biological functions of L1 is scarce. Fungicidal and bactericidal activities have been found (19-21), however, and the term calprotectin has therefore been suggested (9,27). High concentrations of L1 have previously been found in the synovial fluid (SF) from R A patients ( 1 ) . The aim of the present study was to analyze the concentrations of L1 in plasma and SF from patients with R A and osteoarthritis (OA). Comparisons were made with the corresponding concentrations of several acute phase proteins and the differential leukocyte counts. The present study indicates that plasma L1 levels distinguish between an inflammatory and a non-inflammatory disease, and it suggests that L1 in SF may be a marker of joint inflammation.
MATERIALS A N D METHODS Pa tien ts Out- and in-patients at the Department of Rheumatology, The Norwegian Lutheran Hospital, Oslo, were studied. All the patients had swelling of a knee which required relieving synoviocentesis. Forty-one patients had definite or classic R A (22), including 30 females and 11 males, aged 19 to 87 years (median 59), with a disease duration of 3 months to 45 years (median 5 years). Twelve were seropositive for rheumatoid factor (RF), with a titer 1 64 by Waaler test, while 20 were RF positive by the Latex fixation test. Six patients, five females and one male, aged 56 to 76 years (median 64), had O A in one knee as confirmed by X-ray. The O A patients had suffered from knee symptoms for 4 to 25 years (median 8). The patients received a variety of nonsteroidal anti-inflammatory drugs and/or second line anti-rheumatic drugs. SF was obtained from knee joints at the time of synoviocentesis. The exclusion criteria were: Corticosteroid injection into the actual knee joint during the last 3 months, previous osmium acid treatment or surgical synovectomy, secondary arthrosis. suspected bacterial arthritis, knee joint trauma or ongoing systemic infection (anamnesis). Clinical examinations Before joint aspiration, all patients were examined clinically by one of the authors (H.B.B or U . O . ) . Both the amount of swelling and the degree of tenderness, was graded as none (0), slight ( l ) , moderate ( 2 ) and severe (3). Pain in the actual knee joint was estimated by a visual analogue scale. Laboratory investigations The S F was aspirated and placed in a tube with sodium ethylene diamine tetra-acetate (EDTA). Hyaluronidase (PenetraseR), 20 IEiml SF, was added, and the S F was incubated for about 0.5 h before analysis for leukocyte count and differential count. To obtain S F devoid of cells, the fluid was centrifuged at 400 x g for 10 minutes. The supernatant was analyzed for L1, CRP, orosomucoid, haptoglobin, a,-antitrypsin and albumin. S F was frozen at -70°C until analysis of L1.
75
76 H . B . Berntzen et al.
...::
10O.LlOO
-y
... ." Downloaded by [NUS National University of Singapore] at 02:57 21 October 2015
10.000
Scand J Rheumatology 20
...
...
I .
."
I00
RA PLASM
OA
RA
OA
SYNOVIAL FLUID
Fig. 1. The concentrations of L1 in plasma and synovial fluid from rheumatoid arthritis (RA) and osteoarthritis (OA) patients.
The blood samples were analyzed for L1, CRP, orosomucoid, haptoglobin, a,-antitrypsin. albumin and leukocyte counts, as well as differential counting of leukocytes. EDTA-plasma was frozen at - 70°C until analysis of L1. LL in plasma and in SF was analyzed by EIA (Fagerhol e t al, unpublished observations). In brief, wells were coated with rabbit anti-Ll IgG, and L1 in plasma or S F was detected by polyclonal rat ant-L1 and thereafter alkaline phosphatase-conjugated rabbit anti-rat IgG. The coefficient of variation is about 5% within assay and 13% between assays. Normal plasma reference values of L1 (median k 2SD) are 8&880 wg/L (median 440) for females and 15C910 pg/L (median 530) for males. C R P in serum was analyzed by turbidimetry, orosomucoid, haptoglobin and a,-antitrypsin in serum by the use of Behring (Germany) automated nephelometer, and albumin in serum by a colorimetric method using the BioRad (USA) Coomassie Blue reagent. Total leukocyte counts in blood and SF were analyzed in an electronic cell analyzer, and differential counting was performed on May-Griinwald-Giemsa stained smears. Statistics The Mann-Whitney test was used to test for differences between two groups, while KruskalWallis test was used to analyze for differences between multiple groups. The Spearman rank correlation (r,) was used in the correlation analysis, and significance of the correlations was tested by use of Student's t-test. All p values reported were two-tailed. P values < 0.05 were considered significant.
RESULTS Laboratory results L1 levels in plasma and SF showed highly significant differences (p < 0.0001) between the R A and O A patients (Table I and Fig.1). Significant differences (0.001 < p < 0.05) between the two patient groups were also found for the concentrations of orosomucoid, CRP,
L l in plasma and svnovial fluid
Scand J Rheumatology 20
Downloaded by [NUS National University of Singapore] at 02:57 21 October 2015
Table I. The concentrations of LI and acute phase proteins in blood ( B ) and synovial fluid (SF) f r o m rheumatoid arthritis ( R A ) and osteoarthritis (OA)patients (median values with the ranges in parenthesis). Reference values are given f o r the blood analysis. Significant differences between the concentrations in the R A and OA patients are indicated. Laboratory variables
RA patients (n=41)
B-L I I SF-L I I B-alb' SF-alb' B-Oroso' SF-Oroso' B-a I -at4 SF-u,-at' B-CRP' SF-CRP' B-Hapto" SF-Hapto"
9400 ( 98546078) 18156 (1951-375368) 34 (2542) 22 (l(k34) 1.62(0,863.10) 0.99 (0.462.30) 2.1 (1.2-3.7) 1.2 (0.62.5) 33 (0-117) 13 (0-71) 4.I (0.4-7.0) 1.2 (0.1-4.2)
I
kg/L. albumin - g/L. ' Orosomucoid
-
Reference values
OA patients (n=6)
630 (495- 832)"' 895 (29C-2014)"' 40 (3542)' 23 (Zk27) 0.88 (0.661.01 )" 0.49 (0.11-0.68)" 1.5 (1.1-1.5)' 0.8(0.2-1.0)* 2 ((b15)' 2 ((M)" 2.2 (1 x 3 . 3 ) ' 0.3(0.1-1.1)'
g/L. a,-antitrypsin - g/L, ' mg/L,
9"
10
35-50 0.5-1.4 0.8-1.7
< 10 0.3-1.8
' Haptoglobin - g/L.
p
