European Journal of Clinical Investigation (1992) 22,488-493
Tumor necrosis factor alpha is associated w i t h disease activity and the degree of anemia in patients with rheumatoid arthritis G. VREUGDENHIL*t, B. LOWENBERG$, H. G. VAN EIJKS & A. J. G. SWAAKt, *Department of Internal Medicine, Division of Hematology, Sint Radboud University Hospital Nijmegen, The Netherlands; TDepartment of Rheumatology, Dr Daniel den Hoed Clinic, Rotterdam, The Netherlands; $Department of Hematology, University Hospital Dykzigt, Rotterdam, The Netherlands and §Department of Chemical Pathology, Erasmus University, Rotterdam, The Netherlands Received 15 November 1991 and in revised form 14 February 1992; accepted 24 February 1992 Abstract. To elucidate the role of tumor necrosis factor alpha (TNF) in determining anemia of chronic disease (ACD) in rheumatoid arthritis (RA), 24 patients were studied for disease parameters, T N F serum levels and bone marrow for erythroid colony growth and compared with six controls. Serum TNFa was highest in ACD and correlated well with RA disease parameters. Both TNF and other RA disease parameters correlated inversely with degree of anemia. BFUe counts were lower in ACD, correlated positively with Hb and negatively with erythrocyte sedimentation rate (ESR). T N F reduced whereas anti-TNF upregulated in uitro erythroid colony counts. T N F production occurred in similar amounts in bone marrow cultures in the three groups, From these preliminary findings we conclude that ACD in RA correlates with by RA disease activity and that T N F may serve not only as an RA disease marker but also could be one of the factors mediating impaired erythropoiesis in ACD in active RA. Keywords. Anemia, BFUe, erythroid colony growth, Rheumatoid arthritis, Tumor Necrosis Factor alpha (TNFu). Introduction
Anemia is frequently observed in patients with active rheumatoid arthritis (RA) [I]. Several causes of anemia are recognised in RA, e.g., deficiencies of iron [2,3], vitamin B12 [4] and folic acid [5,6]. The most frequent type of anemia in RA, however, is the anemia of chronic disease (ACD) [7]. Many studies have been carried out to examine the pathogenesis of ACD in RA. Decreased iron release from the mononuclear phagocyte system (MPS) [8,9], decreased iron absorption [ 101and decreased erytropoietin responsiveness to the anemia and decreased erythroblast sensitivity [6,1I] have been reported to play a role in the pathogenesis of ACD. More recent investigations have
also addressed the possible role of interleukins as mediators of suppressed erythropoiesis. It was shown that Tumor Necrosis Factor alpha (TNF) was frequently found to be elevated in serum of patients with active disease [12,13] and it may play a pathogenetic role in RA [14]. In addition T N F may possibly have inhibitory effects on erythropoiesis [15,16]. The aim of this study was to examine whether serum T N F levels are related to RA activity, whether ACD is associated with increased serum T N F and whether T N F and anti-TNF affect in uitro erythropoiesis in order to establish the role of T N F in the pathogenesis of ACD in RA. Patients and methods Patients
Bone marrow from 24 (seven male) patients with classical RA according to the revised criteria of the American Rheumatism Association and six normal donors were studied after giving written informed consent: Group I ( n = 6 ) consisted of bone marrow donors (considered as healthy controls). Group 11 ( n = 1 I ) consisted of nonanemic RA patients, group I11 consisted of RA patients with ACD ( n = 13) (for definition see under ‘Laboratory procedures’). Patients who had iron, vitamin B12 or folic acid treatment recently or patients with a present or past ulcer history, hematuria, hypermenorrhoea. positive occult fecal blood test, hemolysis, vitamin B 12 or folic acid deficiency or decreased creatinin clearance were excluded. Overall disease duration was 8 years (3-18), 7 1YO used long-acting antirheumatic drugs (patients using corticosteroids or cytostatic drugs were also excluded) and 82% used nonsteroidal anti-inflammatory drugs. Mean age was 62 years. These characteristics did not differ significantly in the three RA groups. Laboratory procedures
Correspondence: Dr G. Vreugdenhil, Department Internal Medicine, Division of Hematology, University Hospital, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
488
Hemoglobin (Hb, range: 7.4-10.9 mmol 1-I), Hematocrit (Ht, range: 0.36-0.54), reticulocytes, serum iron
RA, TNFa AND ANEMIA
489
Table 1. Erythrocyte parameters, BFUe count, parameters of RA disease activity and TNF in healthy controls and RA patients with and without anemia (ACD). Values expressed as median with range
Hb (mmol I-') Ht (L L-1) reticulocytes (O/OO) BFUe count (per los cells) ESR (MM h-I) CRP (mg L-I) Clqba Rose1 TNF~ (u m1-I)
I controls (n = 6)
I1 Ill non anemic/RA ACD/RA (n= I I ) (n= 13)
8.1 (7'7-8.2) 0.40 (0.37-040) 16 (3-21) 346 (84-396) 12 (8-16) 4 (2-6) 3 (34) 0 0.42 (0.24-0.48)
8.0 (7.7-8.7) 0.38 (0.35-0.42) 10 (1-39) 305 (215-391) 36* (21-65) 22* (2-54) 5 (3-32) 64 (0-512) 0.78 (0.54- 1.68)
6.4f (5.7-7.2) 0.31f (0.28-0.36) 17 (1-30) I89* (12-308) 761 (34-105) 633 ( 10- I0 1 ) I4* (4-75) 16 (0-256) I .68t (0.78-5.58)
IReciprocal Rose titre; 2TNFa in U ml-l x 1667=pg ml-I. Data were compared with controls: * P < 0.05, t P < 0.02, SP < 0.01.
(range: 14-30 pmol I-'), transferrin (range: 44-80 pmol I - ' ) and ferritin (range: 20-150 pg 1 - I ) were assessed by routine laboratory procedures. Erythrocyte sedimentation rate (ESR; < 15 mm h-I) was assessed by the Westergren method, Creactive protein (CRP; < 6 mg I - ] ) was measured by immunodiffusion techniques (Behring Werke, Marburg, Germany), Clq binding assay (Clqba; < 7%) was measured by a method described by Zubler et al. [17].Waaler Rose test was assessed using sensitized sheep erythrocytes; a titre over 1/32 was considered positive [ 181. Serum TNFu was measured immunoradiometrically using monoclonal antibodies against distinct epitopes of TNFcr after coated tube separation (s.a. IRE-M EDGENIX, Fleurus, Belgium) (reference values in 60 control sera: 6.3 pg ml-' or 0.38 U ml-I; range 5-8 pg ml-' or 0.3-0.48 U ml -I). Bone marrow was aspirated after posterior superior iliac crest puncture. Iron content was measured using Perl's Prussian blue staining. A stainable iron content of 2 or more on a semiquantitative scale was considered consistent with ACD [19]. A cell suspension was prepared from 20 ml bone marrow collected in Hank's balanced salt solution (HBSS) with heparin diluted in HBSS and layered over a Ficoll gradient (1.077 g cm2, Nycomed, Oslo, Norway). After centrifugation the mononuclear cells were harvested, washed twice in HBSS and resuspended in HBSS [20]. A cell suspension of lo5cells was added to a mixture of Iscove's modified Dulbecco medium (IMDM), 0.4 ml with 2% methylcellulose, 0.3 ml fetal calf serum, 0.1 ml mixture (containing BSA, transferrin, lecithin, sodium selenite and 2-mercaptoethanol) and 0.015 ml erythropoietin (1 U L- I ) . This volume was divided over four petri-dishes which were
incubated at 37°C and 100% humidity in an environment of 5 % C02 in air. Burst forming units (BFUe; containing 50 or more cells) were counted after 14 days of incubation (standard BFUe count). In group I (n=6), I1 ( n = 5 ) and I11 ( n = 7 ) 100 U ml-' of TNFcr (0.06 U pg-I), 1000 U ml-l of TNFcr (Boehringer Institute, Vienna, Austria) and 1000 U ml-' of anti-TNFcr (0.06 U pg-I) (Boehringer Institute, Vienna, Austria), respectively, were added to the BFUe cultures to evaluate their effects on in vitro erythropoiesis. Next 1 million mononuclear cells were added to a mixture of0.40 ml IMDM, 0.30 ml fetal calf serum and 0.1 ml BFUe mix after which IMDM was added until a volume of I ml. It was then stored in a test tube and incubated at 37°C and 100% humidity in 5% COz air. After 48 h the suspension was centrifugated and the supernatant tested for T N F using the same assay. Statistics
The student-t-test was used for normally distributed data and the Mann Whitney U-test was used for nonparametrical data. Data were correlated using Spearman's coefficient of correlation. A P-value less than 0.05 was considered significant. Results Parameters of erythropoiesis, disease activity, TNF and iron status in the diferent patient groups Erythropoiesis (Table 1). Levels of Hb, Ht and reticulocyte count did not differ between groups I and 11. Negative correlations were found between Hb and ESR (r = -0-78; P < 0.0005), CRP ( r = - 0.44; P
Tumor necrosis factor alpha is associated w i t h disease activity and the degree of anemia in patients with rheumatoid arthritis G. VREUGDENHIL*t, B. LOWENBERG$, H. G. VAN EIJKS & A. J. G. SWAAKt, *Department of Internal Medicine, Division of Hematology, Sint Radboud University Hospital Nijmegen, The Netherlands; TDepartment of Rheumatology, Dr Daniel den Hoed Clinic, Rotterdam, The Netherlands; $Department of Hematology, University Hospital Dykzigt, Rotterdam, The Netherlands and §Department of Chemical Pathology, Erasmus University, Rotterdam, The Netherlands Received 15 November 1991 and in revised form 14 February 1992; accepted 24 February 1992 Abstract. To elucidate the role of tumor necrosis factor alpha (TNF) in determining anemia of chronic disease (ACD) in rheumatoid arthritis (RA), 24 patients were studied for disease parameters, T N F serum levels and bone marrow for erythroid colony growth and compared with six controls. Serum TNFa was highest in ACD and correlated well with RA disease parameters. Both TNF and other RA disease parameters correlated inversely with degree of anemia. BFUe counts were lower in ACD, correlated positively with Hb and negatively with erythrocyte sedimentation rate (ESR). T N F reduced whereas anti-TNF upregulated in uitro erythroid colony counts. T N F production occurred in similar amounts in bone marrow cultures in the three groups, From these preliminary findings we conclude that ACD in RA correlates with by RA disease activity and that T N F may serve not only as an RA disease marker but also could be one of the factors mediating impaired erythropoiesis in ACD in active RA. Keywords. Anemia, BFUe, erythroid colony growth, Rheumatoid arthritis, Tumor Necrosis Factor alpha (TNFu). Introduction
Anemia is frequently observed in patients with active rheumatoid arthritis (RA) [I]. Several causes of anemia are recognised in RA, e.g., deficiencies of iron [2,3], vitamin B12 [4] and folic acid [5,6]. The most frequent type of anemia in RA, however, is the anemia of chronic disease (ACD) [7]. Many studies have been carried out to examine the pathogenesis of ACD in RA. Decreased iron release from the mononuclear phagocyte system (MPS) [8,9], decreased iron absorption [ 101and decreased erytropoietin responsiveness to the anemia and decreased erythroblast sensitivity [6,1I] have been reported to play a role in the pathogenesis of ACD. More recent investigations have
also addressed the possible role of interleukins as mediators of suppressed erythropoiesis. It was shown that Tumor Necrosis Factor alpha (TNF) was frequently found to be elevated in serum of patients with active disease [12,13] and it may play a pathogenetic role in RA [14]. In addition T N F may possibly have inhibitory effects on erythropoiesis [15,16]. The aim of this study was to examine whether serum T N F levels are related to RA activity, whether ACD is associated with increased serum T N F and whether T N F and anti-TNF affect in uitro erythropoiesis in order to establish the role of T N F in the pathogenesis of ACD in RA. Patients and methods Patients
Bone marrow from 24 (seven male) patients with classical RA according to the revised criteria of the American Rheumatism Association and six normal donors were studied after giving written informed consent: Group I ( n = 6 ) consisted of bone marrow donors (considered as healthy controls). Group 11 ( n = 1 I ) consisted of nonanemic RA patients, group I11 consisted of RA patients with ACD ( n = 13) (for definition see under ‘Laboratory procedures’). Patients who had iron, vitamin B12 or folic acid treatment recently or patients with a present or past ulcer history, hematuria, hypermenorrhoea. positive occult fecal blood test, hemolysis, vitamin B 12 or folic acid deficiency or decreased creatinin clearance were excluded. Overall disease duration was 8 years (3-18), 7 1YO used long-acting antirheumatic drugs (patients using corticosteroids or cytostatic drugs were also excluded) and 82% used nonsteroidal anti-inflammatory drugs. Mean age was 62 years. These characteristics did not differ significantly in the three RA groups. Laboratory procedures
Correspondence: Dr G. Vreugdenhil, Department Internal Medicine, Division of Hematology, University Hospital, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
488
Hemoglobin (Hb, range: 7.4-10.9 mmol 1-I), Hematocrit (Ht, range: 0.36-0.54), reticulocytes, serum iron
RA, TNFa AND ANEMIA
489
Table 1. Erythrocyte parameters, BFUe count, parameters of RA disease activity and TNF in healthy controls and RA patients with and without anemia (ACD). Values expressed as median with range
Hb (mmol I-') Ht (L L-1) reticulocytes (O/OO) BFUe count (per los cells) ESR (MM h-I) CRP (mg L-I) Clqba Rose1 TNF~ (u m1-I)
I controls (n = 6)
I1 Ill non anemic/RA ACD/RA (n= I I ) (n= 13)
8.1 (7'7-8.2) 0.40 (0.37-040) 16 (3-21) 346 (84-396) 12 (8-16) 4 (2-6) 3 (34) 0 0.42 (0.24-0.48)
8.0 (7.7-8.7) 0.38 (0.35-0.42) 10 (1-39) 305 (215-391) 36* (21-65) 22* (2-54) 5 (3-32) 64 (0-512) 0.78 (0.54- 1.68)
6.4f (5.7-7.2) 0.31f (0.28-0.36) 17 (1-30) I89* (12-308) 761 (34-105) 633 ( 10- I0 1 ) I4* (4-75) 16 (0-256) I .68t (0.78-5.58)
IReciprocal Rose titre; 2TNFa in U ml-l x 1667=pg ml-I. Data were compared with controls: * P < 0.05, t P < 0.02, SP < 0.01.
(range: 14-30 pmol I-'), transferrin (range: 44-80 pmol I - ' ) and ferritin (range: 20-150 pg 1 - I ) were assessed by routine laboratory procedures. Erythrocyte sedimentation rate (ESR; < 15 mm h-I) was assessed by the Westergren method, Creactive protein (CRP; < 6 mg I - ] ) was measured by immunodiffusion techniques (Behring Werke, Marburg, Germany), Clq binding assay (Clqba; < 7%) was measured by a method described by Zubler et al. [17].Waaler Rose test was assessed using sensitized sheep erythrocytes; a titre over 1/32 was considered positive [ 181. Serum TNFu was measured immunoradiometrically using monoclonal antibodies against distinct epitopes of TNFcr after coated tube separation (s.a. IRE-M EDGENIX, Fleurus, Belgium) (reference values in 60 control sera: 6.3 pg ml-' or 0.38 U ml-I; range 5-8 pg ml-' or 0.3-0.48 U ml -I). Bone marrow was aspirated after posterior superior iliac crest puncture. Iron content was measured using Perl's Prussian blue staining. A stainable iron content of 2 or more on a semiquantitative scale was considered consistent with ACD [19]. A cell suspension was prepared from 20 ml bone marrow collected in Hank's balanced salt solution (HBSS) with heparin diluted in HBSS and layered over a Ficoll gradient (1.077 g cm2, Nycomed, Oslo, Norway). After centrifugation the mononuclear cells were harvested, washed twice in HBSS and resuspended in HBSS [20]. A cell suspension of lo5cells was added to a mixture of Iscove's modified Dulbecco medium (IMDM), 0.4 ml with 2% methylcellulose, 0.3 ml fetal calf serum, 0.1 ml mixture (containing BSA, transferrin, lecithin, sodium selenite and 2-mercaptoethanol) and 0.015 ml erythropoietin (1 U L- I ) . This volume was divided over four petri-dishes which were
incubated at 37°C and 100% humidity in an environment of 5 % C02 in air. Burst forming units (BFUe; containing 50 or more cells) were counted after 14 days of incubation (standard BFUe count). In group I (n=6), I1 ( n = 5 ) and I11 ( n = 7 ) 100 U ml-' of TNFcr (0.06 U pg-I), 1000 U ml-l of TNFcr (Boehringer Institute, Vienna, Austria) and 1000 U ml-' of anti-TNFcr (0.06 U pg-I) (Boehringer Institute, Vienna, Austria), respectively, were added to the BFUe cultures to evaluate their effects on in vitro erythropoiesis. Next 1 million mononuclear cells were added to a mixture of0.40 ml IMDM, 0.30 ml fetal calf serum and 0.1 ml BFUe mix after which IMDM was added until a volume of I ml. It was then stored in a test tube and incubated at 37°C and 100% humidity in 5% COz air. After 48 h the suspension was centrifugated and the supernatant tested for T N F using the same assay. Statistics
The student-t-test was used for normally distributed data and the Mann Whitney U-test was used for nonparametrical data. Data were correlated using Spearman's coefficient of correlation. A P-value less than 0.05 was considered significant. Results Parameters of erythropoiesis, disease activity, TNF and iron status in the diferent patient groups Erythropoiesis (Table 1). Levels of Hb, Ht and reticulocyte count did not differ between groups I and 11. Negative correlations were found between Hb and ESR (r = -0-78; P < 0.0005), CRP ( r = - 0.44; P
