British Journal of Rheumatology 1992;31:675-678
INCREASED SPONTANEOUS RELEASE OF CYTIDINE DEAMINASE BY POLYMORPHONUCLEAR NEUTROPHILS OF PATIENTS WITH ACTIVE SYSTEMIC LUPUS ERYTHEMATOSUS BY C.-L. YU, C.-Y. TSAI*, K.-H. SUN, Y.-S. CHEN, W.-M. LIN, T.-S. LIAO, K.-H. CHENf AND S.-R. WANG Section of Allergy, Immunology and Rheumatology, Department of Medicine, Veterans' General HospitalTaipei; * Institute of Clinical Medicine, tSchool of Medical Technology, National Yang-Ming Medical College; $ Division of Rheumatology, Cathay General Hospital, Taipei, Taiwan, ROC
KEY WORDS: Cytidine deaminase, Systemic lupus erythematosus, Polymorphonuclear neutrophil, A'-Formylmethionyl-leucyl-phenylalanine.
deaminase (CD) is a cytoplasmic enzyme existing in many kinds of cells [1] that catalyses the hydrolytic deamination of cytidine, deoxycytidine and their analogues [2]. The enzyme may provide a salvage pathway for pyrimidine nucleosides produced during the breakdown of nucleic acids [2]. It is claimed that CD may leak out of damaged cells and reflects the turnover rate of local granulocytes (PMN) [1,3]. Several authors reported the increased CD level in the serum of abnormal pregnancy [4] and in the serum and synovial fluid of rheumatoid arthritis [1, 5-7]. In addition, the CD concentration in joint fluid is 20 times more than in serum of the rheumatoid patients [1], suggesting local production of CD at acute inflammatory sites. Systemic lupus erythematosus (SLE) is a classic autoimmune disease characterized by immune-mediated inflammatory reaction in multiple organs. Antineutrophil antibodies could be found in 60% of SLE and would derange the functions of neutrophil and cause neutropenia [8, 9]. In an attempt to understand further the abnormality of SLE PMN, we measured the CD activity in the cytoplasm and in the spontaneous and N - formly - methionyl - leucyl - phenylalanine [FMLP]stimulated culture supernatants of SLE PMN compared with those of normal PMN. We found that the spontaneous release of CD by PMN was significantly higher in active SLE rather than inactive SLE or normal controls.
MATERIALS AND METHODS
CYTIDINE
Patients and controls Twenty-seven patients fulfilling four or more of the 1982 revised American Rheumatism Association criteria for the classification of systemic lupus erythematosus were investigated. Twenty age- and sex-matched normal individuals were the controls. The average age was 26.4 ± 6.6 years in the patient group and 25.6 ± 4.7 years in the control group. Fifteen patients were active and 12 patients were inactive judged by LACC criteria [10]. The mean daily dose of prednisolone was 20.1 ± 8.4 mg in the active SLE group and 8.6 ± 2.2 mg in the inactive group. Four patients in the active group were treated concomitantly with prednisolone and azathioprine (50-100 mg/day). Isolation of mononuclear cells (MNC), polymorphonuclear neutrophils (PMN) and red blood cells (RBC) from peripheral blood Heparinized venous blood obtained from normals or patients was first mixed with a quarter of a volume of 2% dextran solution (mol. wt 500 000) and incubated at 37°C for 20 min. The following procedures for isolation of MNC and PMN have been described in our previous report [11]. The RBC were obtained from the pellet portion of blood after dextran sedimentation. The purity of PMN was greater than 95% as confirmed by Wright's stain.
Submitted 24 June; revised version accepted 11 September 1991. Correspondence to Chia-Li Yu, Section of Allergy, Immunology and Rheumatology, Department of Medicine, Veterans' General Hospital-Taipei, #201 Section 2, Shih-Pai Road, Taipei, Taiwan 11217, Republic of China.
Preparation of culture supernatants and sonicates of different cells 0.36 ml of PMN, MNC or RBC suspension (5.56 x © 1992 British Society for Rheumatology
0263-7103/92/010675 + 04 $08.00/0 675
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 15, 2015
SUMMARY Cytidine deaminase activity (CD) in the neutrophil culture supernatants (PMN SUP) of 27 patients with systemic lupus erythematosus (SLE) was measured using a spectrophotometric method. Compared with the controls (5.449 + 1.358 U/5 x 106 PMN), the CD6activity in the spontaneous culture supernatants of 6PMN was significantly increased in active (10.003 ± 2.637 U/5 x 10 PMN) but not in inactive (5.358 ± 1.624 U/5 x 10 PMN) SLE. However, after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 X 10~7 M), the ratio of enzyme activity between stimulated and spontaneous PMN supernatants was decreased in active SLE (0.794 ± 0.178) compared with normal controls (1.300 ± 0.225). In contrast, the enzyme activity in the cytoplasm of either stimulated or non-stimulated PMN was not different among these three groups. These results suggest that CD of PMN is releasable and can be enhanced by chemotactic factor stimulation in normal PMN. The increased spontaneous release of CD by active SLE PMN is one of the indicators for the disease activity in these patients.
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 10
676
TABLE I COMPARISON OF CYTIDINE DEAMINASE ACTIVITY IN THE CYTOPLASM AND SPONTANEOUS CULTURE SUPERNATANT OF NORMAL PMN, MNC AND RBC
CD activity (units/5 x 10" cells)
Cytoplasm Supernatant
PMN
MNC
RBC
76.18 ±6.45 5.53 ± 1.09
4.27 ±0. 94
INCREASED SPONTANEOUS RELEASE OF CYTIDINE DEAMINASE BY POLYMORPHONUCLEAR NEUTROPHILS OF PATIENTS WITH ACTIVE SYSTEMIC LUPUS ERYTHEMATOSUS BY C.-L. YU, C.-Y. TSAI*, K.-H. SUN, Y.-S. CHEN, W.-M. LIN, T.-S. LIAO, K.-H. CHENf AND S.-R. WANG Section of Allergy, Immunology and Rheumatology, Department of Medicine, Veterans' General HospitalTaipei; * Institute of Clinical Medicine, tSchool of Medical Technology, National Yang-Ming Medical College; $ Division of Rheumatology, Cathay General Hospital, Taipei, Taiwan, ROC
KEY WORDS: Cytidine deaminase, Systemic lupus erythematosus, Polymorphonuclear neutrophil, A'-Formylmethionyl-leucyl-phenylalanine.
deaminase (CD) is a cytoplasmic enzyme existing in many kinds of cells [1] that catalyses the hydrolytic deamination of cytidine, deoxycytidine and their analogues [2]. The enzyme may provide a salvage pathway for pyrimidine nucleosides produced during the breakdown of nucleic acids [2]. It is claimed that CD may leak out of damaged cells and reflects the turnover rate of local granulocytes (PMN) [1,3]. Several authors reported the increased CD level in the serum of abnormal pregnancy [4] and in the serum and synovial fluid of rheumatoid arthritis [1, 5-7]. In addition, the CD concentration in joint fluid is 20 times more than in serum of the rheumatoid patients [1], suggesting local production of CD at acute inflammatory sites. Systemic lupus erythematosus (SLE) is a classic autoimmune disease characterized by immune-mediated inflammatory reaction in multiple organs. Antineutrophil antibodies could be found in 60% of SLE and would derange the functions of neutrophil and cause neutropenia [8, 9]. In an attempt to understand further the abnormality of SLE PMN, we measured the CD activity in the cytoplasm and in the spontaneous and N - formly - methionyl - leucyl - phenylalanine [FMLP]stimulated culture supernatants of SLE PMN compared with those of normal PMN. We found that the spontaneous release of CD by PMN was significantly higher in active SLE rather than inactive SLE or normal controls.
MATERIALS AND METHODS
CYTIDINE
Patients and controls Twenty-seven patients fulfilling four or more of the 1982 revised American Rheumatism Association criteria for the classification of systemic lupus erythematosus were investigated. Twenty age- and sex-matched normal individuals were the controls. The average age was 26.4 ± 6.6 years in the patient group and 25.6 ± 4.7 years in the control group. Fifteen patients were active and 12 patients were inactive judged by LACC criteria [10]. The mean daily dose of prednisolone was 20.1 ± 8.4 mg in the active SLE group and 8.6 ± 2.2 mg in the inactive group. Four patients in the active group were treated concomitantly with prednisolone and azathioprine (50-100 mg/day). Isolation of mononuclear cells (MNC), polymorphonuclear neutrophils (PMN) and red blood cells (RBC) from peripheral blood Heparinized venous blood obtained from normals or patients was first mixed with a quarter of a volume of 2% dextran solution (mol. wt 500 000) and incubated at 37°C for 20 min. The following procedures for isolation of MNC and PMN have been described in our previous report [11]. The RBC were obtained from the pellet portion of blood after dextran sedimentation. The purity of PMN was greater than 95% as confirmed by Wright's stain.
Submitted 24 June; revised version accepted 11 September 1991. Correspondence to Chia-Li Yu, Section of Allergy, Immunology and Rheumatology, Department of Medicine, Veterans' General Hospital-Taipei, #201 Section 2, Shih-Pai Road, Taipei, Taiwan 11217, Republic of China.
Preparation of culture supernatants and sonicates of different cells 0.36 ml of PMN, MNC or RBC suspension (5.56 x © 1992 British Society for Rheumatology
0263-7103/92/010675 + 04 $08.00/0 675
Downloaded from http://rheumatology.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 15, 2015
SUMMARY Cytidine deaminase activity (CD) in the neutrophil culture supernatants (PMN SUP) of 27 patients with systemic lupus erythematosus (SLE) was measured using a spectrophotometric method. Compared with the controls (5.449 + 1.358 U/5 x 106 PMN), the CD6activity in the spontaneous culture supernatants of 6PMN was significantly increased in active (10.003 ± 2.637 U/5 x 10 PMN) but not in inactive (5.358 ± 1.624 U/5 x 10 PMN) SLE. However, after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 X 10~7 M), the ratio of enzyme activity between stimulated and spontaneous PMN supernatants was decreased in active SLE (0.794 ± 0.178) compared with normal controls (1.300 ± 0.225). In contrast, the enzyme activity in the cytoplasm of either stimulated or non-stimulated PMN was not different among these three groups. These results suggest that CD of PMN is releasable and can be enhanced by chemotactic factor stimulation in normal PMN. The increased spontaneous release of CD by active SLE PMN is one of the indicators for the disease activity in these patients.
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 10
676
TABLE I COMPARISON OF CYTIDINE DEAMINASE ACTIVITY IN THE CYTOPLASM AND SPONTANEOUS CULTURE SUPERNATANT OF NORMAL PMN, MNC AND RBC
CD activity (units/5 x 10" cells)
Cytoplasm Supernatant
PMN
MNC
RBC
76.18 ±6.45 5.53 ± 1.09
4.27 ±0. 94
