Humans Tohoku J. Exp. Med., 2002, 198, 125-132 Sulfite is Generated from PAPS by Activated Neutrophils Hex Mirsumasut, Foote Ora, Kazvnte Ixrucet, Yortaxt Kanexo, ‘Taxasur Kororwa, Kazur Urxt, Yosnrro Tsxapa and Yosnmsa Nos ‘The Third Department of Internal Medicine, Gunma University School of Medicine, Macbaski 371-8511 Morsvuasm, H., Ora, F., Ixevomt, K., Kanexo, Y., Kuronwa, T., Uzxt, K., ‘Tsuxapa, Y. and Noma, Y. Sulfite is Generated from PAPS by Activated Neutrophils. Tohoku J. Exp. Med., 2002, 198 (2), 125-132——- We previously reported that neutrophils produce sulfite in response to stimulation with ipopolysaccharide, and sulfite production is dependent on inorganic sulfate contained in culture media. Microorganisms such as yeast assimilate sulfate, during which process sulfite is generated by reduction of 3’-phosphoadenosine 5’-phosphosulfate (PAPS), an activated sulfate donor. However, little is known about how sulfite is produced in mammalian cells. Tn the current study, we demonstrated that chlorate, a specific inhibitor for PAPS synthesis, significantly suppressed production of sulfite by activated neutrophils obtained from rat peritoneal cavity that had been injected with glycogen to induce inflammation. Addition of excess amounts of PAPS could partially overcome the inhibitory effect of chlorate. Moreover, sulfite production from PAPS was clearly demon- strated in the cytosolic fraction of activated neutrophils. ‘These findings strongly suggest that sulfite is generated, at least in part, from PAPS by activated neutrophils, —— sulfite; neutrophil; PAPS © 2002 Tohoku University Medical Press are exposed to substantial sulfite loading tests in healthy volunteers amounts of sulfite via the inhalation of sulfur dioxide in the atmosphere, the metabolism of endogenous sulfur-containing amino acids, or by the intake of various foods that contain sulfiting agents as antioxidants and antimi- erobials (Gunnison 1981; Shore et al. 1987). Recent studies have shown that human serum contains sulfite at a concentration of 0-10 Bf (Ji et al. 1995; Kajiyama et al. 2000). Oral Received June 3, 2002; revision accepted for publication October 28, 2002. revealed that the blood sulfite level peaks at 100-200 «M within 30minutes after ingestion and returns to normal levels within a few hours without any adverse reactions (Ji et al. 1995). This rapid clearance of sulfite appears, at least in part, to be due to sulfite oxidase, a mitochon- drial enzyme contained in most mammalian tissues (Garrett et al. 1995). This enzyme oxi- dizes sulfite to sulfate, which is immediately Address for reprints: Dr. Yoshihisa Nojima, The Third Department of Internal Medicine, Gunma Univer- sity School of Medicine, Macbashi 371-8511, Japan, e-mail: ynojima@ med.gunma-u.ac.jp 125126 HE Mitsuhashi et al excreted into urine. A rare congenital deficiency in sulfite oxidase has been identified in humans, and these patients develop severe neurological abnormalities leading to death at an early stage of life (Johnson and Wadman 1995). Brain damage is thought to occur as a result of the accumulation of sulfite at toxic levels causing marked neuronal loss and demyelination (Johnson and Wadman 1995). Thus, the intracellular sulfite should be maintained within adequate levels in normal individuals. Yang et al. (1996) reported that sulfite is spontaneously excreted from rabbit neutrophils. We recently demonstrated that activated human neutrophils produce sulfite and serum sulfite levels were significantly inereased in a rat model of sepsis induced by injection with bacte- rial endotoxin, lipopolysaccharide (LPS) (Mitsuhashi et al. 1998). Given its potent antioxidant and antimicrobial activities, we proposed a putative role of endogenous sulfite in host defense and inflammation. Microorgan- isms such as yeast assimilate inorganic sulfate, during which process sulfite is generated by reduction of 3’-Phosphoadenosine 5’- phosphosulfate (PAPS), an activated sulfate donor (Munakata et al. 1981; Li et al. 1987; Schwenn et al. 1988; Gutierrez-Mareos etal. 1996). We have shown that withdrawal of sulfate from culture media resulted in the fail- ure of sulfite production by activated neutro- phils (Mitsuhashi et al. 1998), Although this observation suggested that sulfate is also used as a souree of sulfite in mammalian cells, little is known about the precise mechanism. In the present report, we firstly demonstrate that PAPS works as a substrate in sulfite production by activated neutrophils. MATERIAL AND METHODS Materials Sodium sulfite, sodium sulfate and mono- bromobimane were purchased from Funakoshi (Tokyo). PAPS, glycogen, and sodium chlor- ate were obtained from Sigma Chemical Co. (St. Lovis, MO, USA). Sodium borohydrate and Ficall-Hypaque were from Wako (Tokyo). Dextran T-500 was from Pharmacia Biotech (Uppsala, Sweden). Rat neutrophil isolation from peripheral blood and peritoneal cavity Male Wister rats, specific pathogen free (Charles River Japan, Atsugi), were used. Blood was collected from the abdominal aorta of pentobarbital anesthetized rats, and neutro- phils were purified by dextran sedimentation, hypotonic lysis of erythrocytes, and centrifuged over Ficall-Hypaque. Cells were washed sev- eral times and resuspended in Hanks’ balanced salt solution (HBSS) and kept on an ice-bath until use. To obtain peritoneal neutrophils, 20 ml of 1% glycogen in sterile saline was adminis- tered intraperitoneally to rats (Miles et al. 1995). Two hours later, rats were sacrificed and cells infiltrated into the peritoneal cavity were collected. The purity of neutrophils obtained by this method was more than 97% as assessed by Wright staining, and the cell viabil- ity was more than 90%. Cells were washed several times and resuspended in HBSS, and kept on an ice-bath until use. Preparation of neutrophil cytosolic fractions ‘Neutrophils (1 10* cells/ml) were disrupt- ed in HBSS by sonication on ice and then centrifuged at 14.000 g for 20 minutes at 4°C. Supernatant was used as the cytosol fraction, Determination of sulfite concentration Determination of sulfite in samples was previously deseribed (Mitsuhashi et al. 1998). Standardized solution of sulfite or samples (100 ul) were mixed with 701 of 70mM mono- bromobimane in acetonitrile. After incubation for 10 minutes at 42°C, the mixture was added with 50 l of 1.5M perchloric acid followed by vortex-mixing, The protein precipitate was removed by centrifugation at 12400g for 8Sulfite Production from PAPS by Neutrophils 127 minutes, Twenty microliters of the neutralized supernatant was injected onto HPLC (Hitachi 655-11 system). The column was equilibrat- ed with methanol: acetic acid: water (5.00: 0.25 : 94.75, by volume, pH3.4) and developed via a methanol gradient in acetic acid: water (0.25 : 94.15, by volume) at a flow rate of 0.8 mi/minutes as follows: 0 to 5 minutes, 30 ml/ liter; 5 to 13 minutes, 30 to 350 ml/liter; 13 to 23 minutes, 350 to 620 ml/liter; 23 to 24 mi utes, 620 to 1000 ml/liter; 24 to 29 minutes, 1000 mi/liter; 29 to 30 minutes, 1000 to 30 ml/ liter; and 30 to 34 minutes, 30 ml/liter. Sulfite-bimane was detected by excitation at 390 nm and emission at 472 nm using cut-off filter and eluted at 45 ml/liter of methanol. Standard solutions of sulfite were prepared fresh for each assay by dissolving sodium sulfite in HBSS, and a calibration curve was obtained by ‘measuring the relative fluorescence intensity of sulfite-bimane. Statistical analysis All values are expressed as meants.p. Individual group means were compared with the Student’s t-test. RESULT Neutrophils obtained from peripheral blood and the peritoneal cavity were incubated in HBSS containing sulfate at 0.4 mM at 37°C for 60 minutes at a density of 210° cells/200 yel/well in 96-well flat-bottom culture plates. Culture media were collected at the indicated periods, and the sulfite concentration was deter- mined by the HPLC method, As compared with resting peripheral blood neutrophils, per- itoneal neutrophils released significantly hi amounts of sulfite into culture media (Fig. 1). Sulfite production by peritoneal neutrophils was 2.7241.01 (mean-ts.p.) nmol/10’ cells at the 60 minutes incubation time point, which was 6-fold higher than that by peripheral blood neutrophils (0.44+0.14 nmol/10" cells). Gly- cogen chemically peritoneal induces - 4 = 2 3 é mo B41 £ _ Ts 2 oe a o 30 60 Incubation Time (min) Fig 1. Sulfite production from activated neutro- phils, Cizeulating blood neutrophils (closed cireles) and neutrophils obtained from the inflamed peritoneal cavity (closed squares) ‘were incubated in sulfate-contained HBSS for the indicated periods and suite production ‘was measured by reversed-phase HPLC. Each value represents the mean-+s.p. of four independent experiments inflammation, thereby providing a source of activated neutrophils. Thus, the present finding supports our previous idea (Mitsuhashi et al. 1998) that activated neutrophils generate sulfite at the site of inflammation. In the following experiments, peritoneal neutrophils were used to study the mechanism of sulfite production. As previously reported (Mitsuhashi et al. 1998), sulfite production by activated neutrophils is dependent on inorganic sulfate contained in the culture media, In accordance with this, complete withdrawal of sulfate from the culture media also markedly reduced sulfite release by peritoneal neutrophils (Fig. 2, compare columns 1 and 2). This sug- gests that sulfate is utilized to generate sulfite in activated neutrophils. In yeast and enter- bacteria, inorganic sulfate is assimilated by a sequential enzymatic cascade, in which PAPS is produced as an intermediate substrate (Klaassen and Boles 1997). PAPS synthesis is clearly dependent on the availability of in- organic sulfate, and PAPS sulpho-transferase