Introduction

Acetylsalicyclic acid (ASA), also called aspirin, is an established and widely used drug, mainly due to its broad therapeutic spectrum with anti-inflammatory, antipyretic, and analgesic effects by predominantly inhibiting cyclooxygenase (COX) activity, which contributes to the conversion of arachidonic acid to prostaglandins (PGs).This suppression of PG synthesis by COX inhibition is the major effect of ASA and its metabolite, salicylate [1, 2]. Recently, ASA has also been shown to protect allograft function and survival as well as to reduce complications in transplant recipients [3–5], but the exact mechanism is not clear.

The acute and chronic rejection of allografts and xenografts is closely related to innate immune processes, including those mediated by granulocytes and macrophages [6, 7]. In addition to their phagocytotic, killing, and pro- and/or anti-inflammatory activities, macrophages signal adaptive responses through a variety of cytokines, co-stimulatory molecules, and antigen presentation [8, 9]. Macrophages can perform nonopsonic and FcγR- and CR3-mediated opsonic phagocytosis to efficiently eradicate pathogens [6]. Moreover, the nonopsonic phagocytosis of macrophages plays a major role in the rejection response to allografts [10]. Some reports described the effect of ASA on the functions of macrophages, e.g. opsonic phagocytosis [11] and nitric oxide [12]. However, the effect of ASA on the nonopsonic phagocytosis and immunogenicity of macrophages has not yet been studied. The present study, therefore, aimed to investigate the effect of ASA on the cell numbers, phenotype, nonopsonic phagocytosis, and immunogenicity of macrophages. The results of the present study showed that ASA significantly decreased cell numbers, nonopsonic phagocytosis, and immunogenicity as well as changing the phenotype of macrophages. Our data offered basic information which may provide new insights for the application of ASA in patients with allografts or autoimmune diseases.

Materials and methods

Animals and reagents

Eight-to-ten-week-old BALB/c (H-2d) and C57BL/6 (B6; H-2b) mice were purchased from Beijing University Experimental Animal Center (Beijing, China). All mice were maintained in a specific pathogen-free facility and were housed in microisolator cages containing sterilized feed, autoclaved bedding, and water. All experimental manipulations were undertaken in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals.

The culture medium used in the present study was RPMI 1640 (Hyclone, Logan, UT, USA) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 10 mM HEPES, and 50 μM 2-ME (Sigma, St. Louis, MO, USA). ASA was purchased from Sigma Chemical Co (St Louis, USA). Mitomycin C (C15H18N4O5) was obtained from Kyowa Hakko Co, Ltd. (Tokyo, Japan). [3H]Thymidine was purchased from the China Institute of Atomic Energy (Beijing, China).

The following monoclonal antibodies (mAbs) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA): phycoerythrin (PE)-conjugated anti-mouse F4/80 mAb (BM8), fluorescein isothiocyanate (FITC)-conjugated anti-CD80 mAb (16-10A1; IgG2b), anti-CD47mAb (MIAP301), and anti-I-Ad mAb (39-10-8). Rat anti-mouse FcR mAb (2.4G2, IgG2b) was produced by 2.4G2 hybridoma (ATCC, Rockville, MD, USA) in our laboratory.

ASA treatment

BALB/c mice were divided into three groups for in vivo studies: control group, low dose ASA-treated group (6 mg/kg), and high dose ASA-treated group (60 mg/kg). ASA (Sigma) was dissolved in DMSO (5 M) and then diluted with normal saline (NS) and administered intraperitoneally at the dose of 6 or 60 mg/kg/day for 1 week. Control group mice were injected with NS (with <0.4% DMSO as the experiment group) only. For in vitro studies, macrophages were treated with different doses of ASA (0, 0.5,1, 2, or 5 mM) and the control buffer (with 0.4% DMSO) as indicated in the figure legends.

Preparation of peritoneal exudate macrophages (PEMs)

Resident peritoneal cells were obtained from the peritoneal exudates of mice as described previously [13, 14]. Briefly, the peritoneal exudate cells were washed twice with cold Hanks’ solution, adjusted to 5 × 106 cells/ml in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) and cultured in 6-well plates (Costar, Cambridge, MA, USA) pretreated with 2% gelatin (Sigma) for 3–4 h at 37°C and 5% CO2. The non-adherent cells were removed by washing with warm RPMI1640 medium. The adherent cells were harvested with 5 mM EDTA (Sigma) in ice-cold PBS (pH 7.2) and re-adjusted to 3 × 105 cells/ml per well into 24-well plates. The cell viability was determined by trypan blue exclusion. The macrophage purity was analyzed by a two-photon microscope (TPM) LSM510 (Zeiss, Wetzlar, Germany) and flow cytometry (FCM) (Becton–Dickinson, Mountain View, CA, USA), using a mouse macrophage marker F4/80, as reported previously [14, 15].

Preparation of bone-marrow-derived macrophages (BMMs)

BMMs were prepared as described previously [16], whereby these macrophages were produced by M-CSF stimulation of bone marrow cells for a total of 7 days. Briefly, BMM were obtained from specific-pathogen-free BALB/c mice (8–10 weeks old). Mice were killed by cervical dislocation and both femurs were dissected free of adherent tissue. The bones were trimmed at each end, and the marrow was flushed out with RPMI 1640 medium. Cells were suspended by pipetting and washed by centrifugation. Red blood cells were lysed and the cell suspension was washed and filtered to remove debris. The initial adherence step in petri dishes removed bone marrow stromal cells and any mature resident bone marrow macrophages. Removal of non-adherent cells at this point allowed progenitor cells to expand and differentiate under the influence of M-CSF. Cell suspensions were then washed and plated in 24-well tissue culture plates at a concentration of 106 cells per well in complete RPMI 1640 medium (10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 10 mM HEPES, and 50 μM 2-ME) containing the indicated concentrations of ASA in the presence of M-CSF (100 ng/ml). Each concentration of drug was tested in triplicate wells. The cell monolayer was then incubated at 37°C in 5% CO2 for 7 days, with the medium changed on day 4. BMMs were obtained after 7 days in culture in the presence of M-CSF. These BMMs could be detected by the expression of F4/80 molecules, and were used for later functional or phenotypic assays.

Immunofluorescence staining and FCM

Aliquots of 2 × 105 mouse macrophages (PEMs or BMMs) were washed once with FACS buffer [PBS, pH 7.2, containing 0.1% NaN3 and 0.5% bovine serum albumin (BSA)]. For two-color staining, cells were stained with PE-labeled anti-mouse F4/80 mAb versus FITC-labeled CD80 (16-10A1), I-Ad (39-10-8), CD86 (GL1), CD47 (MIAP301), or the non-specific staining control mAb, respectively [17]. Non-specific FcR binding was blocked by anti-mouse FcRmAb (clone 2.4G2). At least 10,000 cells were assayed by two-color FCM using a FASCalibur flow cytometry (Becton–Dickinson), and data were analyzed with CellQuest software (Becton–Dickinson). Non-viable cells were excluded using the vital nucleic acid stain propidium iodide (PI). The percentage of cells stained with a particular reagent or reagents was determined by subtracting the percentage of cells stained non-specifically with the isotype control mAb from the percentage stained in the same dot-plot region with the anti-mouse mAbs. Certain molecule expression levels were determined as the median fluorescence intensity (MFI) of the cells positively stained with the specific mAb. The cell size of F4/80+ cells was determined by the analysis of the forward scatter side with gating on F4/80+ cells.

Phagocytosis of xenogeneic chicken red blood cells (cRBCs) by mouse macrophages

A single-cell suspension of cRBCs was freshly obtained. Following two washes with PBS, 1 × 107 cells/ml cRBCs in PBS were labeled with 5.0 μM 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Inc., Eugene, OR, USA) for 15 min at 37°C [18]. These cells were then washed thoroughly and suspended in RPMI 1640 without serum at the required concentration [6]. Cell viability was usually more than 95% as determined by trypan blue exclusion. PEMs treated in vivo were obtained from mice administered intraperitoneally with or without ASA for 1 week. In vitro treated macrophages (PEMs or BMMs) were pre-incubated with or without ASA at the concentrations and times indicated in the figure legends. These ASA-treated PEMs treated both in vivo and in vitro were co-incubated with CFSE-labeled cRBCs at a ratio of 1:10 for 2–2.5 h at 37°C and 5% CO2 in 24-well plates (Costar) that had been pre-treated with 2% gelatin (Sigma) [19]. After 2–2.5 h, un-ingested cRBCs were removed by aspirating supernatants and adding cold PBS to stop phagocytosis. Cells were then gently washed three times with cold PBS. Adherent cells were harvested with 5 mM EDTA (Sigma) in ice-cold PBS (pH 7.2) and re-adjusted. Cells were blocked with anti-mouse FcγR mAb (clone 2.4G2) and stained with PE-conjugated anti-F4/80 mAb (BM8; eBioscience, San Diego, CA, USA). After washing with cold FACS buffer three times, the phagocytosis percentages of F4/80+ gate cells were determined using FCM (BD Biosciences, San Diego, CA, USA). Forward and side scatter gates were set to exclude debris. Data were analyzed using Cell Quest software (BD Biosciences) [20]. Meanwhile, for the phagocytosis assay by TPM, 24-well plates (Costar) had been preset with cover glasses [17]. The cover glasses were washed once and adherent cells were blocked with anti-mouse FcγR mAb (clone 2.4G2) and stained with PE-conjugated anti-F4/80 mAb (BM8). Three-channel images were taken with a two-photon laser scanning microscope (LSM510, Zeiss). Individual macrophages were isolated from Z stacks with the extract region feature and further analyzed using the ortho and gallery displays of the LSM510 imaging software.

Delayed-type hypersensitivity (DTH)

Sensitized C57BL/6 effector T cells were generated by immunization with allogeneic BALB/c splenocytes. Ten days after immunization, C57BL/6 CD4+ T cells were enriched using the negative selecting MACS kit for CD4+ T lymphocytes (BD Biosciences Pharmingen). BALB/c PEMs were used as stimulator cells. Sensitized C57BL/6 effector CD4+ T cells and allogeneic macrophage stimulators (5 × 105 cells each) treated with or without ASA both in vivo and in vitro were co-injected intradermally into the pinna of naïve C57BL/6 mice. The changes in ear thickness were measured using an engineer’s micrometer at 24 or 48 h after challenge [21]. The ear thickness change was calculated by subtracting the thickness of the ear before injection from the thickness of the same ear after injection.

Allogeneic mixed leukocyte reactions (MLRs)

Murine splenocytes were prepared using the sterile technique as described previously [22]. Cells were suspended in RPMI 1640 medium supplemented with 10% (v/v) mouse serum, 2 mM l-glutamine, 0.1 mM non-essential amino acids (GibcolBrl), 1 mM sodium pyruvate, 10 U/ml penicillin and 10 μg/ml streptomycin, 10 mM HEPES buffer (GibcolBrl), and 10 μM 2-mercaptoethanol [23]. Triplicate wells containing 1 × 105 responders with 1 × 105 of allogeneic macrophage stimulators (PEMs pretreated with 50 μg/ml mitomycin C) in a total volume of 0.2 ml of medium were incubated in U-bottomed 96-well microplates (Costar) at 37°C in 5% CO2. Cells were cultured at 37°C and 5% CO2 for 4 days. [3H]Thymidine (0.5 μCi; radioactivity 185 GBq/mmol; Atomic Energy Research Establishment, China) was added for the last 18 h [24]. Cells were harvested onto glass fiber filters with an automatic cell harvester (Tomtec, Toku, Finland). Samples were assayed in a Liquid Scintillation Analyzer (Beckmon, Fullerton, CA, USA). Values are expressed as counts per minute (cpm) from triplicate wells and are the results after subtracting cpms from wells in the absence of stimulator cells [25].

Cell viability analysis (MTT assay)

MTT assay was also used to measure the number of viable cells. After incubation of murine PEMs with different concentrations (0.5, 1, 2, and 5 mM) of ASA, cells were incubated with 20 μl MTT solution (5 mg/ml) per well for another 4 h at 37°C. After removal of the supernatant, DMSO 150 μl was added and shaken for 5 min until the crystals were dissolved. The optical density (OD) value of each well was recorded using spectrophotometry with a microplate reader (Bio-Rad Smartspec, Model 450, USA) at 490 nm. The reading is directly proportional to cell viability and results are expressed as percentage of cell viability with respect to control absorbance (100% cell viability). The same procedure was repeated three times and the OD value was calculated for statistical analysis.

Statistical analysis

All experiments were performed at least three times and in triplicate. The data are given as the mean ± SD. Data were analyzed using Student’s unpaired t test. P < 0.05 was considered significant.

Results

ASA significantly altered cell number and phenotype of mouse PEMs in vivo

The cell numbers and phenotype of macrophages were firstly evaluated 1 week after BALB/c mice were treated with ASA (6 or 60 mg/kg/day, respectively). As shown in Fig. 1a, a significant decrease in cell numbers of PEMs in mice treated with the high dose (60 mg/kg/day) of ASA for 1 week was observed compared with control-treated mice (P < 0.01). The expression of co-stimulatory molecules on antigen presenting cells (APCs) is crucial in determining the nature and extent of the immune response. We therefore studied the expressions of major histocompatibility complex (MHC)-II molecules (I-Ad), co-stimulatory molecules (CD80), and CD47 on murine F4/80+ PEMs. When the phenotype of PEMs were detected using two-color FCM, significantly lower percentages of F4/80+ PEMs expressed MHC II and CD47 molecules in ASA-treated mice than those in control mice (Fig. 1 b, c), while the expression of CD80 did not change significantly after ASA treatment. The expression levels of CD47 and I-Ad molecules on F4/80+ cells were significantly decreased after ASA treatment (P < 0.05; Fig. 1d). Thus, alteration of cell numbers and phenotype of F4/80+ PEMs occurred in the ASA-treated mice.

Fig. 1
figure 1

The cell numbers and phenotype of PEMs from mice treated with or without ASA for 1 week. a Cell numbers of PEMs from mice treated with or without ASA. b Phenotype characteristics of murine F4/80+ PEMs. The murine PEMs were stained with PE-labeled anti-F4/80 mAb versus FITC-labeled I-Ad, CD80, CD86, or CD47 mAbs. Ten thousand F4/80+ cells were analyzed by FCM. A representative of the phenotype of murine F4/80+ PEMs was assessed by FCM. The black filled histograms represent the non-specific mAb staining and open histograms represent the indicated mAb staining. c The percentages of I-Ad, CD80, and CD47-positive cells in murine F4/80+ PEMs treated with or without ASA. d The levels of I-Ad, CD80, or CD47 molecules in murine F4/80+ PEMs treated with or without ASA as determined by two-color FCM. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding groups. More than six mice in each group were examined

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ASA significantly decreased the nonopsonic phagocytosis and immunogenicity of mouse PEMs in vivo

To determine whether or not ASA has the ability to alter the functions of PEMs in vivo, we performed nonopsonic phagocytosis and DTH assays. Phagocytosis represents an early and crucial event in triggering host defenses against invading pathogens. Mouse F4/80+ PEMs separated from ASA-treated mice were co-cultured with CFSE-labeled cRBCs for 2–2.5 h and the phagocytic ability of macrophages was investigated using FCM. Mouse F4/80+ PEMs in control mice have the ability to phagocytize cRBCs efficiently, as reported previously (Fig. 2). However, macrophages separated from mice treated with ASA showed significantly decreased nonopsonic phagocytosis compared with the control mice (P < 0.01; Fig. 2b).

Fig. 2
figure 2

Significantly decreased phagocytosis and immunogenicity of PEMs from mice treated with ASA for 1 week. a One representative showing BALB/c F4/80+ PEM phagocytosis of xenogeneic cRBCs as detected by FCM. BALB/c PEMs were stained with PE-labeled anti-F4/80+ mAb, whereas cRBCs were labeled with CFSE. BALB/c PEMs were co-cultured with cRBCs as described in the “Materials and methods” section. b The mean percentages of phagocytosis cells in F4/80+ PEMs isolated from mice treated with or without ASA. c DTH responses induced by BALB/c PEMs from ASA treated or control mice were detected at 24 h after injection into the pinna of naïve C57BL/6 mice. Six mice in each group were assayed and three independent experiments were performed. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding groups

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We then observed the immunogenicity of macrophages of ASA-treated BALB/c mice in vivo by using an allo-DTH assay. Sensitized C57 BL/6 CD4+ T cells were co-injected intradermally with allogeneic PEMs separated from ASA-treated BALB/c mice into the pinna of naïve C57BL/6 mice. The changes in ear thickness were measured at 24 h after challenge. As shown in Fig. 2c, significant DTH responses were observed if sensitized C57BL/6 T cells were stimulated by allogeneic BALB/c PEMs. However, PEMs from BALB/c mice treated with 6 or 60 mg/kg/day ASA for 1 week induced significantly lower DTH responses of sensitized C57BL/6 T cells did than those of control mice (P < 0.05, P < 0.01, respectively; Fig. 2c).

ASA inhibited the differentiation of bone marrow stem cells into macrophages in vitro

Mononuclear phagocytes develop in the bone marrow, circulate in the blood as monocytes, and are resident in all tissues of the body as macrophages. To explore whether the alteration of macrophages induced by ASA in vivo was a result of changes in macrophage differentiation, we examined the effect of ASA on the differentiation of bone marrow stem cells into macrophages in vitro. As shown in Fig. 3, ASA markedly reduced the number of BMMs, indicating that ASA may block the differentiation of bone marrow stem cells into macrophages. In addition, the phenotypic analysis of BMMs by two-color FCM revealed that ASA-treated BMMs expressed lower levels of CD80, CD47, and I-Ad as compared to control (Fig. 3b).

Fig. 3
figure 3

The direct effect of ASA on the differentiation of BMMs in vitro. a ASA blocked the differentiation of macrophages from bone marrow cells in the presence of M-CSF. a control; b DMSO control; c 1 mM ASA; d 5 mM ASA. b ASA decreased the cell numbers of macrophages derived from bone marrow cells in the presence of M-CSF. c ASA changed the phenotype of BMMs. BMMs were stained with PE-labeled anti-F4/80 mAb versus FITC-labeled I-Ad, CD80, and CD47 mAb, respectively. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding groups

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To evaluate the function of these BMMs, we performed nonopsonic phagocytosis by FCM. These BMMs treated with or without ASA in vitro were co-cultured with CFSE-labeled cRBCs for 2–2.5 h, and the phagocytic ability of macrophages was investigated using two-color FCM. As shown in Fig 3c, ASA-treated BMMs exhibited a significant lower phagocytic ability as compared to control BMMs (P < 0.01).

Effect of ASA on the phenotype and nonopsonic phagocytosis of mouse PEMs in vitro

To investigate the potential direct effect of low (1 mM) and high (5 mM) doses of ASA on the phenotypic characteristics of F4/80+ PEMs in vitro, we studied the expressions of MHC-II molecules (I-Ad), co-stimulatory molecules (CD80), and CD47 on murine F4/80+ PEMs. In vitro treatment with ASA in both low and high doses for 24 h significantly down-regulated the expression of CD80, CD47 and I-Ad on macrophages (Fig. 4).

Fig. 4
figure 4

The direct effect of ASA on the phenotype of murine PEMs in vitro. Murine PEMs were cultured with or without ASA (1 and 5 mM) for 24 h in vitro, and then stained with PE-labeled anti-F4/80 mAb versus FITC-labeled I-Ad, CD80, or CD47 mAb. Ten thousand F4/80+ cells were analyzed by FCM. a One representative of the phenotype of murine F4/80+ PEMs was assessed by FCM. The black filled histograms represent the non-specific mAb staining and open histograms represent the indicated mAb staining. b The percentages of I-Ad, CD80, and CD47-positive cells in murine F4/80+ PEMs treated with or without ASA. C, The levels of I-Ad, CD80, or CD47 molecules in murine F4/80+ PEMs treated with or without ASA as determined by two-color FCM. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding groups

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Murine PEMs treated by ASA in vitro were co-cultured with CFSE-labeled cRBCs for 2–2.5 h and the phagocytic ability of macrophages was investigated using FCM. As shown in Fig. 5, the incubation of murine F4/80+ PEMs with ASA for 12 h in concentrations of 0.5, 1, and 2 mM did not exert significant effects on nonopsonic phagocytosis but exerted a significant inhibitory effect on nonopsonic phagocytosis at the concentration of 5 mM (Fig. 5b). However, incubation of murine F4/80+ PEMs with ASA for 24 h at concentrations of 0.5, 1, 2, and 5 mM exerted significant inhibitory effects on the nonopsonic phagocytic activity of murine PEMs (Fig. 5c).

Fig. 5
figure 5

The direct effect of ASA on the phagocytosis of PEMs treated with ASA. Mouse PEMs were cultured with different levels of ASA (0.5, 1, 2, or 5 mM) in vitro. The phagocytosis was detected by FCM. Medium and DMSO indicate control. a One representative of the phagocytosis of murine F4/80+ PEMs (treated with ASA for 24 h) to cRBCs as determined by FCM. The mean percentage of phagocytosis cells in F4/80+ PEMs treated with ASA for 12 h and 24 h is summarized in (b) and (c), respectively. Data are one representative of three independent experiments. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding groups

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The direct effect of ASA on the immunogenicity of murine PEMs

The direct effect of ASA on the immunogenicity to allogeneic T cells of BALB/c PEMs was determined in BALB/c PEMs treated with low (1 mM) and high (5 mM) doses of ASA or without ASA in vitro. BALB/c PEMs treated with ASA showed significantly decreased immunogenicity to allogeneic T cells compared with control, as evaluated in a MLR assay (P < 0.001; Fig. 6a). To further determine the immunogenicity of ASA-treated murine PEMs by DTH assay, sensitized C57BL/6 CD4+ T cells were co-injected intradermally with allogeneic BALB/c PEMs (treated with or without ASA in vitro) into the pinna of naïve C57BL/6 mice. As shown in Fig. 6b, significant DTH responses were observed if sensitized C57BL/6 CD4+ T cells were stimulated by allogeneic BALB/c PEMs. However, BALB/c PEMs treated with low and high doses of ASA in vitro induced significantly lower DTH responses of sensitized C57BL/6 T cells than did those of control mice (P < 0.01; Fig. 6b).

Fig. 6
figure 6

The direct effect of ASA on the immunogenicity of murine PEMs in vitro. a The immune responses of C57BL/6 CD4+ T cells stimulated by allogeneic BALB/c PEMs treated with or without ASA in vitro as determined by MLR. b DTH response induced by allogeneic BALB/c PEMs treated with or without ASA was detected in C57BL/6 mice. Data are presented as mean ± SD. **P < 0.01; ***P < 0.001 compared with the corresponding groups

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Discussion

The effect of ASA on macrophages in transplantation recipients is of interest since macrophages are key components in the immune response, performing different functions including phagocytosis and antigen presentation [8]. Resident macrophages are generally considered to be derived from circulating monocytes [26, 27]. In the present study, the effect of ASA on cell numbers, phenotype, and functions were detected in resident murine PEMs in both in vivo and in vitro assays. These observations indicated that ASA decreased cell numbers and functions and changed the phenotype expressions of PEMs. The decrease in cell numbers may be caused by the effect of ASA on macrophage viability and/or blocking the differentiation of macrophages from monocytes or bone marrow cells (BMCs). Our results showed that various concentrations of ASA used in the present study did not affect the viability of macrophages, based on MTT assay. In addition, ASA-treated murine BMCs produced a significantly lower number of F4/80+ macrophages than did ASA-untreated murine BMCs. These data support the possibility that the decrease in macrophage cell numbers in ASA-treated mice is due, at least in part, to the deficient differentiation of macrophages from BMCs, though other possibilities were not excluded.

Macrophages are a major component of the mononuclear phagocyte system that consists of closely related cells of bone marrow origin, including blood monocytes and tissue macrophages [28]. Phagocytosis by macrophages is an essential step and a part of innate immunity for protection against foreign pathogens, microorganisms, or dead cells [29]. ASA has previously been shown to enhance the phagocytosis of IgG-opsonized sheep RBCs by murine PEMs [30]. Nevertheless, other authors showed no significant effect of ASA on opsonic phagocytosis of Candida albicans by murine tumor-associated and peritoneal macrophages [31]. However, the effect of ASA on nonopsonic phagocytosis of xenogeneic cells (cRBCs) by macrophages is unknown. Therefore, our study focused particularly on the effect of ASA on the nonopsonic phagocytosis of xenogeneic cells by macrophages, which plays an important role in the rejection response to allografts [10]. Murine F4/80+ PEMs treated with ASA both in vitro and in vivo have significantly lower phagocytosis capacity against cRBCs than those of control groups as assayed by TPM or FCM. Consistently with the ASA-treated murine F4/80+ PEMs, ASA-treated BMMs showed significantly lower phagocytosis capacity against cRBCs than those of control groups. A significantly decreased phagocytosis capability of host macrophages exerted by ASA may be related to its immunosuppressive effect protecting allograft function and survival as well as reducing complications in transplant recipients, as previously reported [3–5, 32]. ASA might enhance the opsonic phagocytosis of macrophages [30], though some reports showed inconsistent data [31]. The reasons for the different effects of ASA on opsonic and nonopsonic phagocytosis are not clear at this point. It may be due to the different molecular networks involved in opsonic and nonopsonic phagocytosis, which need to be determined in the near future.

In order to gain more insight into the effect of ASA on the functional activity of macrophages, we also studied the effect of ASA on the immunogenicity of murine PEMs both in vitro and in vivo by MLR and DTH assays, respectively. Our results indicated that murine PEMs treated with a high dose of ASA (5 mM) showed significantly decreased immunogenicity to allogeneic T cells of murine PEMs as compared to control.Significant DTH responses were consistently observed if sensitized C57BL/6 T cells were stimulated by allogeneic BALB/c PEMs. However, BALB/c PEMs treated with ASA both in vivo and in vitro induced significantly less DTH responses of sensitized T C57BL/6 cells than did those of control mice, which may be related to their low levels of MHC-II and co-stimulatory molecule expressions, because many different molecules and signaling networks cooperate to activate and regulate T cells when they encounter a display of immunogenic peptides by APCs. It should be noted that the ASA doses used for our present in vivo and in vitro studies are not comparable and that the doses of ASA used in vitro and in vivo are working via different mechanisms. Previous studies in vitro have indicated that ASA as low as 1 mM inhibited NF-κB activity [33] whereas only a dose of ASA 600 mg/kg/day could inhibit NF-κB in vivo [34]. The related molecular mechanisms should be clarified in the future.

In summary, ASA decreases cell numbers, phagocytosis and immunogenicity of macrophages as well as changing the phenotype expressions of macrophages. ASA has the ability to block the differentiation of macrophages. These data may offer important implications for the clinical application of ASA to prevent graft rejection and treat individuals with autoimmune diseases.