Expression and activity of hydrogen sulfide generating enzymes in murine macrophages stimulated with lipopolysaccharide and interferon-γ

Murine macrophages of the J774A.1 line are hydrogen sulphide-producing cells with the primary role of γ-cystathionase (CTH) and secondary role of 3-mercaptopyruvate sulfurtransferase (limited by cysteine availability) and with a negligible role of cystathionine β-synthase (CBS) in H2S generation. J774A.1 cells stimulation with lipopolysaccharide (LPS) or interferon-gamma (IFNγ) resulted in decreased H2S levels after 24 h of incubation; however, they were restored to the control level after 48 h. Negligible CBS expression and activity in J774A.1 cells can result in homocysteine availability for CTH-catalyzed, H2S-generating reactions. This was supported by an increased CTH expression (IFNγ, 24 h and 48 h, and LPS, 48 h) and activity (24 h, LPS) in the stimulated cells. The results confirm the suggested feedback regulation between CBS and CTH.


Introduction
Methionine is metabolized to homocysteine (HCys) to produce cysteine, which is a substrate of two enzymes-cystathionine β-synthase (CBS) and γ-cystathionase (CTH)involved in endogenous production of H 2 S (Scheme 1) [1]. In cells with low CBS expression, H 2 S can be alternatively generated by CTH-catalyzed condensation reaction between cysteine and HCys or between two molecules of HCys (Scheme 1) [2].
Another endogenous source of H 2 S is cysteine transamination by cysteine aminotransferase (CAT) and subsequent 3-mercaptopyruvate (3MP) conversion by 3-mercaptopyruvate sulfurtransferase (MPST) [3]. 3MP provides sulfur to the active-site cysteine residue of MPST to produce persulfide, which releases H 2 S in the presence of thioredoxin (Trx), abundant in cells. Another physiological reducing disulfide is dihydrolipoic acid (DHLA), cofactor for mitochondrial α-ketacid dehydrogenases. Trx and DHLA release H 2 S from persulfide provided by 3MP at the active site of MPST [4]. The expression of CBS, CTH and MPST are tissue-specific.
Hydrogen sulfide (H 2 S) plays an important role in inflammation [5] but there is no clear consensus as to its precise role in inflammatory signaling. Macrophages in general play important roles in the initiation and progression of many chronic inflammatory diseases. Li et al. [6] observed biphasic effects of H 2 S on inflammatory signals in murine lipopolysaccharide (LPS)-treated macrophages, where low H 2 S levels inhibited LPS-induced synthesis of PGE 2 , NO, IL-1β, IL-6 and NF-κB activity, but higher NaHS concentrations promoted synthesis of pro-inflammatory mediators.
LPS-a potent activator of macrophages, and interferongamma (IFNγ)-the principal macrophage-activating factor, 1 3 was used to stimulate murine macrophages of the J774A.1 cell line. H 2 S measured by the zinc acetate-trapping method [7] was used to compare the H 2 S level in the control, and LPS-and IFNγ-stimulated cells after 24 and 48 h of incubation. The expression and the activity of hydrogen sulfidegenerating enzymes, i.e. CBS, MPST, CTH, and TST, were investigated in J774A.1 cells. The results indicate an important role of CTH in the generation of hydrogen sulfide in J774A.1 cells with low activity of the transsulfuration pathway catalyzed by the CBS/CTH enzymatic system. They also confirm a negative feedback regulation between CBS and CSE [8].
The studies presented in this paper were designed to determine the activity and expression of hydrogen sulfide generating enzymes, i.e.: cystathionine β-synthase (CBS), γ-cystathionase (CTH) and MPST in murine macrophages stimulated with LPS and IFN-γ.

Cell culture
Mouse BALB/c monocyte macrophage of the J774A.1 cell line (ATCC, Manassas, VA, USA) were grown in DMEM medium supplemented with 10% FCS, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), in sterile, plastic culture flasks (BD Falcon, Franklin Lakes, NJ, USA), at 37 °C, in a humidified atmosphere containing 5% CO 2 . For experiments, the cells were cultured in DMEM medium supplemented with 5% FCS and antibiotics (as above) at 37 °C, in a humidified atmosphere containing 5% CO 2 , either on sterile, plastic Petri dishes (100 mm in diameter) or on sterile, plastic, 24-well, flat-bottom plates. J774A.1 cells were stimulated for 24 h and 48 h with LPS (100 ng/5 × 10 5 cells/ml; Sigma Aldrich, St. Louis, MO, USA) or with recombinant IFNγ (100 U/5 × 10 5 cells/ml; PeproTech, London, UK). When necessary, after a certain time of stimulation, dry cell pellets were frozen and stored at − 80 °C prior to RNA isolation.

Isolation of total RNA
Total RNA was extracted with Trizol reagent (Lab-Empire, Poland) following the manufacturer's instructions and RNA quantity and integrity were verified by agarose gel electrophoresis. Isolated RNA was stored at − 80 °C and was used for RT-PCR.
For CTH, after an initial 5 min denaturation at 94 °C, amplification was performed under the following conditions: 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 2 min for 35 cycles, with a final incubation at 72 °C for 8 min.
For CBS, after an initial 5 min denaturation at 94 °C, amplification was performed under the following conditions: β-actin was used as an internal standard to normalize mRNA abundance. PCR products were analyzed by 2.0% agarose gel electrophoresis and imaged by UVI-KS 4000i/ ImagePC (Syngen Biotech, Poland).

Detection of H 2 S
H 2 S produced during the incubation of the cell culture with H 2 S-releasing compounds was trapped as zinc sulfide in the zinc agarose layer according to [7]. The standard curve was linear at the concentration range of 0-250 µM with a correlation coefficient of 0.994.

Cell homogenization
Murine macrophage cells (line J774A.1) (3.5-5 × 10 6 cells/ pellet) were resuspended in 0.1 M phosphate buffer pH 7.5, in the proportion 1 × 10 6 cells/0.07 ml of the buffer, sonicated 3 × 5 s at 4 °C (Bandelin Sonoplus GM 70). After centrifugation at 1600 × g for 10 min, the supernatant was used for the determination of protein concentration, sulfane sulfur levels and the activity of MPST, CTH and rhodanese. For reverse phase high performance liquid chromatography (RP-HPLC) analyses, the cells were resuspended in 0.1 ml 10% PCA/1 mM BPDS. The sediment was separated by centrifugation at 1400 × g for 10 min, and the supernatant was stored at − 80 °C until analysis.

MPST activity
The MPST activity was assayed according to the method of Valentine and Frankelfeld 1974 [13] with some modification as described by Wróbel et al. [14] and Bronowicka-Adamska et al. [15]. The enzyme activity was expressed as nmoles of pyruvate produced during 1 min incubation at 37 °C/1 mg of protein.

CTH activity
The cystathionase activity was determined by Matsuo and Greenberg [16] with modifications described by Czubak et al. [17]. The difference between the initial value of absorbance (before adding LDH) and the lowest value (after adding LDH) corresponded to the amount of alpha-ketobutyrate formed in the course of the cystathionase reaction. The cystathionase activity was expressed as nmoles of α-ketobutyrate formed during 1 min incubation at 37 °C/1 mg of protein.

CBS activity
The activity of CBS was examined in cell homogenates in the presence of dl-homoserine as a substrate after 15 min incubation at 37 °C according to the description by Bronowicka-Adamska et al. [18]. The level of cystathionine was determined using the HPLC method described by Bronowicka-Adamska et al. [19]. The CBS activity was expressed as pmoles of cystathionine formed during 1 min incubation at 37 °C/1 mg of protein.

Sulfane sulfur level
Sulfane sulfur was determined by the method of Wood [20] following the procedure described by Bronowicka-Adamska et al. [15]. The method is based on cold cyanolysis and colorimetric detection of ferric thiocyanate complex ion. The sulfane sulfur level was expressed as nmoles of SCN − produced/1 mg of protein.

Protein level
Protein concentration was determined by the method described by Lowry et al. [21] using crystalline bovine serum albumin as a standard.

RP-HPLC (reverse phase high performance liquid chromatography)
The levels of cystathionine in the incubation mixtures were determined using the RP-HPLC method Dominik et al. [22] with modifications [19].

Statistical analysis
All the results from at least three independent experiments, 15-25 individual determinations, were expressed as mean ± SD. The significance of the differences between the controls and investigated samples were calculated using is a nonparametric Mann-Whitney U test (p < 0.05).

The release of TNFα and IL-6 by J774A.1 cells stimulated with IFNγ or LPS
After stimulation with both factors, macrophages released proinflammatory mediators. The effects of LPS stimulation was weaker than IFNγ (Table 2).

H 2 S levels in control, and LPS and IFNγ-stimulated J774A.1 cells
The level of hydrogen sulphide was significantly lower after 24 h in case of both factors (Fig. 1). It was restored to the control levels after 48 h of incubation.

The expression of MPST, CTH, CBS and TST in murine macrophage cells (J774A.1 cells)
After 24 h incubation the control cells, not stimulated (Fig. 2a, line 1), express γ-cystathionase (CTH) and MPST, two H 2 S-forming enzymes. Low TST expression and only trace expression of cystathionine β-synthase (CBS) was found. Similarly after 48 h (Fig. 2a, line 4) with the exception of higher expression of TST, as compare to 24 h, and lower expression of CTH.
CBS expression did not change significantly in the LPSstimulated cells and it seemed even to drop in the IFNγstimulated cells (Fig. 2a, b). The expression of MPST significantly decreased during incubation with 100 U IFNγ and 100 ng/ml LPS. IFNγ, in turn, up-regulated CTH expression in J774A.1 cells after both 24 h and 48 h stimulation, in comparison to the control cells (Fig. 2a, b).

The activity of MPST, CTH, TST and the level of sulfane sulfur in J774A.1 cells after stimulation
CBS activity in both the control and stimulated cells was undetectable. The activity of MPST (24 h) and rhodanese (24 h and 48 h) was significantly decreased after incubation with IFNγ and LPS after 48 h, in comparison to the control cells (Table 3A, B). Significantly increased CTH activity was determined after 24 h and 48 h stimulation with LPS (Table 3A, B) but in case of IFNγ it was below the detection limit after 24 h or significantly decreased after 48 h, as compare to control values. A homeostasis of the level of sulfane sulfur was observed-it remains unchanged independently of the compound used and time of incubation (Table 3A, B).

Discussion
Hydrogen sulfide (H 2 S) has been identified as a regulator of inflammatory responses [5].
The results have confirmed the expression of CTH and MPST in J774A.1 cells suggesting that macrophages are hydrogen sulphide producing cells. CBS seems to be of a low importance for H 2 S formation in J774A.1 cells.
Low CBS expression can result in HCys availability for CTH-catalyzed H 2 S generating reaction [2,23] (Scheme 1). The IFNγ-stimulated production of NO was detected to result in a decrease in CBS activity [24] and it is attributed to a reduced rate of the transsulfuration pathway, CBS/CTH dependent, converting majority of HCys (Scheme 1). In the IFNγ-stimulated cells, an increased expression of CTH (Fig. 2a, b) after 24 h is correlated with a decreased -as compared to control-level of H 2 S. This can result from significantly decreased activity of CTH and TST observed after both 24 h and 48 h of incubation  (Table 3A, B), the activity of which is regulated through a redox-switch regulatory mechanism [25]. However, higher expression of CTH after 48 h, in comparison to 24 h, resulted in an increased level of hydrogen sulfide (Figs. 1, 2a, b).
In case of LPS, a higher production of H 2 S after 48 h, as compare to 24 h, is also correlated with higher increase in CTH expression after both IFNγ and LPS stimulation. An increased expression of CTH and H 2 S production in the macrophages stimulated with LPS was earlier reported   [26]. A decreased expression and activity of MPST after 24 h (Fig. 2a, b; Table 3A) limits the amount of cysteine converted by this enzyme and make it more available for H 2 S-generating reactions catalyzed by CTH (Scheme 1). Interestingly, the expected increased level of HCys in J774A.1 cells with low CBS expression and activity, is associated with a high CTH expression. A decreased level of H 2 S after 24 h stimulation with both IFNγ and LPS was also associated with an increased CTH expression. These observations may confirm the suggested effect of HCys (increased due to low CBS level) and H 2 S on CTH expression, which is upregulated by high levels of HCys and low H 2 S levels and a negative feedback regulation between CBS and CTH [8]. Increased HCys level together with the elevated level of CTH (IFNγ, 48 h) can result in cysteine sucking into reactions with HCys and its lower availability for GSH synthesis. Decreased GSH levels result in an increased level of reactive oxygen species which are, in turn, involved in the redox regulation of immune cells [27]. H 2 S can be regarded an O ⋅− 2 scavenger-it reacts with superoxide radical anion (Scheme 1). H 2 S is a reducing agent and a weak acid with approximately 4:1 HS − /H 2 S ratio at physiological pH [28]. HS − , as opposed to H 2 S, can reduce the metal center of cytochrome c and lead to production of O ⋅− 2 from molecular oxygen [29]. On the other hand, HS − /H 2 S can reduce Cu 2+ to Cu 1+ and enhance O ⋅− 2 scavenging activity of the copper-zinc SOD (CuZnSOD) [29,30]. Sulfide is a potent reversible inhibitor of myeloperoxidase activity [31]. It has also been shown that HCys can induce Trx-1 expression in human monocytes [32] and in this way potentiate the antioxidant protection of cells (Scheme 1). Thus, in cells, H 2 S can act as a pro-or antioxidant, depending on the H 2 S/HS − ratio.
In stimulated cells, hydrogen sulphide can react with nitric oxide [33], what can result in decreased H 2 S levels, especially pronounced after 24 h, and particularly in the IFNγ-stimulated cells with a massive amount of proinflammatory mediators released (Table 1). On the other hand, H 2 S might also exert anti-inflammatory effects by inhibiting NO production [11]. H 2 S oxidation results in persulfides and thiosulfate formation [34], substrates of mitochondrial rhodanese. It catalyzes the transfer of sulfane sulfur atom from thiosulfate to various acceptors (e.g. GSH), leading to H 2 S, and allows for recycling H 2 S without consuming additional cysteine. However, it seems this does not occur due to its significantly decreased activity after 48 h stimulation with both IFNγ and LPS (Table 3A, B). On the other hand, a homeostasis of sulfane sulfur level was observed (Table 3A, B).

Conclusions
J774A.1 macrophage cells are hydrogen sulphide-producing cells with CTH as the main enzyme and MPST playing a supporting role. A low CBS expression and non-detectable CBS activity in the IFNγ-stimulated cells can result in HCys availability for CTH-catalyzed, H 2 S-generating reactions (Scheme 1). A decreased level of H 2 S after 24 h stimulation with both IFNγ and LPS associated with an increased CTH expression can confirm CTH upregulation by a high level of HCys (due to low CBS expression) and low H 2 S and a negative feedback regulation between CBS and CTH.