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* Department of Laboratory Sciences, School of Health Sciences, Faculty of Medicine, Kanazawa University, Kanazawa 920-0942, Japan and
Department of Pediatrics and Angiogenesis and Vascular Development, Kanazawa University Graduate School of Medical Science, 920-8641, Japan
Abstract
Monocytes play key roles both in innate and adaptive antigen-specific immunity and they constitute critical components of the immune responses. Although most of the monocyte-derived cytokines exhibit proinflammatory functions in vivo, heme oxygenase-1 (HO-1), an inducible heme-degrading enzyme, exerts potent anti-inflammatory effect through production of carbon monoxide and bilirubin. We compared HO-1 production by monocytes in vivo in various acute inflammatory illnesses and in normal controls. Freshly isolated monocytes produced little HO-1 as detected by immunohistochemistry, but it was rapidly induced in vitro upon stimulation. HO-1 production by monocytes was selective because it was not induced in other leukocyte populations, including granulocytes and lymphocytes. Monocytes from acute inflammatory illnesses, such as Kawasaki disease and acute infectious diseases, viral or bacterial, produced significant levels of HO-1, as detected by flow cytometry, immunohistochemistry, and reverse transcription polymerase chain reaction. Quantitative analysis of HO-1 mRNA expression by real-time polymerase chain reaction revealed that monocytes from controls exhibited low, but significant levels of HO-1 mRNA, indicating that circulating monocytes produce HO-1 constantly, in response to basal level of oxidative stress encountered daily. Significantly elevated HO-1 mRNA levels seen in acute inflammatory illnesses suggest that monocyte HO-1 production serve as potent anti-inflammatory agent to control excessive cell or tissue injury in the presence of oxidative stress and cytokinemia.
Key Words: inflammation • heme oxygenase • oxidative stress
Monocytes play pivotal roles in various stages of self-defense, including phagocytosis of opsonized pathogens, digestion and processing of foreign antigens, antigen presentation in association with class II molecules and, finally, release of several inflammatory effector molecules. All of these functions constitute critical components of the immune responses (1, 2). Although these proinflammatory functions of monocytes contribute significantly in eradicating invading pathogens in cases of acute infectious diseases, excess and persistent monocyte activation may lead to irreversible damage to tissues and organs. Monocyte activation syndrome, which is frequently encountered in association with rheumatic diseases, is characterized by extreme cytokinemia, organ dysfunctions, bone marrow failure, and systemic inflammatory reactions (3, 4). Excessive production of monocyte-derived cytokines also is associated with several other chronic inflammatory illnesses. Interleukin-6 or tumor necrosis factor-
are representative of such molecules, and recent reports indicate that direct inhibition of their functions can be an effective therapeutic option in some cases of severe inflammatory illnesses (5–7). It is natural, therefore, to assume that these cells possess common mechanisms to counteract inflammatory functions and regulate the production of proinflammatory cytokines and protect cells from various oxidative stresses.
An inducible form of heme oxygenase (HO), HO-1, is one of the likely candidate molecules for such purposes (8, 9). HO-1 is rapidly induced upon stimulation and under various oxidative stresses. Its primary function is to degrade heme into carbon monoxide (CO), biliverdin, and free iron. In addition to its role as a key enzyme in heme degradation, it is recently reported that HO-1 functions as potent anti-inflammatory, anti-oxidative agent through its production of bilirubin and CO (10–12). CO not only acts on vascular smooth muscles to dilate the blood vessels, but it also acts on cellular metabolism and counteracts proinflammatory cytokine cascades. Lack of HO-1, therefore, may lead to persistent inflammatory state, in addition to chronic anemia due to impaired iron reutilization.
We recently experienced a case of human HO-1 deficiency (13). The clinical feature of the patient was characterized by sustained intravascular hemolysis, signs of chronic severe inflammation, and renal tubular dysfunction. Pathological examination revealed the presence of diffuse endothelial injury of the glomerular capillary and damages to renal proximal tubular epithelium (14). In addition to these peculiar findings, the patient exhibited abnormally high concentration of serum haptoglobin–hemoglobin complex, hyperlipidemia, extreme thrombocytosis, and leukocytosis, in association with numerous fragmented erythrocytes within the peripheral blood. It was strongly indicated that cellular injury and dysfunction of vascular endothelium, renal tubular epithelium, and monocyte/macrophage are responsible for the wide spectrum of symptoms observed in the HO-1 deficient patient.
In this study, we compared the levels of HO-1 production by peripheral blood monocytes and different leukocyte subpopulations. We next attempted to elucidate the functional relevance of HO-1 production by monocytes in various acute inflammatory illnesses of children.
Materials and Methods
Subjects.
Peripheral blood samples were obtained from normal volunteers to determine in vitro HO-1 production by different cell populations. To compare HO-1 production by monocytes in various acute inflammatory illnesses, blood samples were obtained from patients with Kawasaki disease, acute bacterial infection, acute infectious mononucleosis, other viral infections, and normal control children. Patients with Kawasaki disease presented with characteristic symptoms and satisfied the clinical criteria as described in the literature (15). Acute infectious mononucleosis was confirmed by typical patterns of EBV VCA IgG and EBNA antibody titers and characteristic increase of activated CD8+ T lymphocytes within the peripheral blood. Other viral infections included adenovirus, measles, varicella, and exanthema subidum. Adenovirus infection was confirmed by rapid enzyme diagnostic kit (16). The latter three diseases were diagnosed clinically based on the characteristic symptoms. Bacterial infections were confirmed by positive C-reactive protein, increased number of circulating leukocytes, and isolation of causative bacteria from the suspected infectious foci. These included pneumonia, acute bronchitis, meningitis, and urinary tract infections. All samples were used after informed consents were obtained from the patients or the guardians.
Cell Preparation.
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by Ficoll/Hypaque density gradient centrifugation. For HO-1 mRNA analysis, purified lymphocyte subpopulations were obtained from PBMCs as follows. To prepare monocyte-enriched fraction, T cells were depleted from PBMCs by rosetting with 2-aminoethylisouronium bromide-treated sheep erythrocytes. Monocytes were further purified from the monocyte-enriched fraction by plastic adherence as described before (17). More than 90% of the plastic adherent cells were CD14+ monocytes as determined by a flow cytometry. Lymphocyte subpopulations were separated into CD4+ T cells, CD8+ T cells, CD16+ natural killer cells, and CD20+ B cells by an electronic cell sorting using Coulter Epics Elite (Coulter Electronics, Hialeah, FL) (18). The purity of each cell population was as high as 99.5%, as determined by a flow cytometry of the recovered cell fractions.
Cell Culture.
Cells were suspended at 106/ml in RPMI1640 culture medium containing, 10% fetal calf serum, 25 mmol/l HEPES, 5 x 10-5 mol/l 2-mercapto-ethanol, 100 U/ml penicillin, and 10 µg/ml gentamycin. All cell cultures were performed at 37°C with 5% CO2. To compare the levels of HO-1 production by monocytes and lymphocyte subpopulations, PBMCs were cultured for 8 hr with 100 µmol/l hemin. To determine HO-1 mRNA expression, fractionated leukocyte subpopulations were cultured alone or in the presence of 100 µmol/l hemin, 10 µmol/l sodium arsenite, or 10 µmol/l cadmium chloride for 2 hr.
Reagents.
MAbs used in this study are as follows. FITC-conjugated anti-CD8 mAb was from Pharmingen International (San Diego, CA). FITC-conjugated anti-CD16 and anti-CD14 mAbs were obtained from Ortho Clinical Diagnostics Co. (Tokyo, Japan). PE-conjugated anti-CD4 mAb was the product of DAKO Japan (Kyoto, Japan). Hemin was the product of Sigma. Anti-HO-1 rabbit antiserum (SPA-896) was from StressGen Biotechnologies Corp. (Victoria, Canada). Anti-rat HO-1 mAb was kindly provided by Dr. M. Suematsu (Keio University, Tokyo). It is known to cross react with human HO-1 (19). Goat anti-rabbit antibody and anti-mouse antibody conjugated with alkaline phosphatase polymer (EnVision Labeled Polymer, Alkaline Phosphatase) was obtained from DAKO Corp. (Carpinteria, CA). Fast Red TR salt and Naphtol As-MX phosphate were the products of Sigma.
Flow Cytometry.
Intracellular HO-1 expression was determined by a flow cytometry after following procedures. The cultured cells were washed in phosphate-buffered saline (PBS) twice and fixed in 1% paraformaldehyde in PBS for 20 min at room temperature. They were further washed in PBS twice and permealized in 0.2% saponin in PBS with 3% fetal calf serum and 0.1% sodium azide (saponin buffer) for 20 min at room temperature. After washing once in saponin buffer, the cells were incubated with mouse anti-HO-1 mAb or with control isotype-matched IgG antibody for 20 min on ice. After washing, these cells were further incubated with FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates Inc., Birmingham, AL) at optimum dilution for 15 min on ice. Monocyte and lymphocyte regions were gated separately and HO-1 expression by each cell population was evaluated independently by Cytoron Absolute flow cytometer (Ortho Diagnostic Systems, Tokyo, Japan).
Immunohistochemistry.
For immunohistochemistry, the cultured cells were washed in PBS twice and cytospin preparations were made, air-dried, fixed in acetone and rinsed in Tris buffer. After blocking with normal goat serum, the slides were stained with appropriate dilutions of anti-HO-1 rabbit antiserum for 1 hr at room temperature. After washing three times in Tris buffer, alkaline phosphatase-conjugated EnVision was reacted for 30 min at room temperature. Alkaline phosphatase activity was visualized using Fast Red TR salt and Naphtol As-MX phosphate (14).
HO-1 mRNA Expression by Mononuclear Cell Subpopulations.
The cultured lymphocyte subpopulations and monocytes were washed in PBS twice and RNA was isolated with a standard acid guanidine phenol chloroform method. Reverse transcription was performed with random hexamer primer (Takara Shuzo Co., Tokyo Japan) and RAV-2 reverse transcriptase (Takara Shuzo). Polymerase chain reaction (PCR) amplification of the entire open reading frame of the HO-1 gene was performed with primers HO-1 5N sense (5'-CTCCCCTCGAGCGTCCTC-3') and HO-1 3N antisense (5'-CCTTCAGTGCCCACGGTAA-3'). The PCR conditions were 95°C for 30 sec, 55°C for 30 sec, and 72°C for 90 sec, for 35 cycles (13). As controls, ß-actin mRNA expression was examined simultaneously.
Real-Time PCR with a Fluorogenic Probe.
The upstream and downstream sequences PCR primers for the HO-1 real-time PCR are 5'-TGAGGAACTTTCAGAAGGGCC-3' and 5'-TGTTGCGCTCAATCCCTCC-3', respectively (Funakoshi, Co. Ltd. Tokyo, Japan). A fluorogenic probe (5'-CGGCTTCAAGCTGGTGATGGCC-3') with a sequence located between the PCR primers was synthesized by PE Applied Biosystems Japan Co. (Tokyo, Japan). The PCR reaction was performed using the TaqMan PCR kit (PE Applied Biosystems Japan) as previously described (13). Briefly, either 250 ng of DNA from each cultured cells stimulated by hemin was added to a PCR mixture containing 10 mM Tris (pH 8.3). 50 mM KCl, 10 mM EDTA, 5.5 mM MgCl2, 100 µM dATP, dCTP, dGTP, and dTTP, 0.6 µM each primer, 0.2 µM fluorogenic probe, and 1.25 U of AmpliTaq Gold for 10 min at 95°C, 45 to 50 cycles of 15 sec at 95°C and 1 min at 62°C were performed by a model 7700 sequence Detector (PE Applied Biosystem). Real-time fluorescence measurement was taken, and a threshold cycle (Ct) value for each sample was calculated by determining the point at which the fluorescence exceeded a threshold limit (10 times the standard deviation of baseline). For a positive control, a plasmid that contained HO-1 gene was constructed from pGEM-T vector (Promega, Madison, WI). A standard graph of the Ct values obtained from serially diluted pGEM-HO-1 was constructed. The Ct values from samples were plotted on the standard curve, and the copy number was calculated automatically by Sequence Detector version 1.6 (PE Applied Biosystem Japan), a software package for data analysis. Each sample was tested in duplicate, and the mean of the two values was shown as the copy number of the sample. Samples were defined as negative if the Ct values exceeded 50 cycles. GAPDH mRNA was quantitated simultaneously, and the HO-1/GAPDH ratio was calculated for each sample.
Results
HO-1 Production by PBMCs.
After culture with 100 µmol/l of hemin for 8 hr, HO-1 production was observed selectively within monocyte populations as shown by immunohistochemistry. Granulocytes or lymphocytes did not produce detectable level of HO-1 (Fig. 1A and B
). This was confirmed by a flow cytometry. Lymphocyte and monocyte regions were gated separately and HO-1 production by these distinct cell populations were compared (Fig. 1C). Virtually all monocytes produced significant levels of HO-1 upon stimulation. In contrast, only a small fraction of lymphocytes produced detectable, but very low level of HO-1.
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). Different types of oxidative stresses, including hemin, sodium arsenite, and cadmium chloride showed essentially similar data.
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) and in acute Kawasaki disease (Fig. 3D–F). Immunohistochemistry of the cytospin preparations revealed that PBMCs from normal controls expressed little HO-1 in the cytoplasm (Fig. 3A), whereas a fraction of PBMCs from acute Kawasaki disease expressed detectable levels of HO-1 (Fig. 3D). A small portion of these cells expressed significantly increased level of HO-1 (arrow). Morphologically, these HO-1 producing cells are monocytes. Flowcytometric analysis clearly showed that both lymphocytes and monocytes from normal control did not express detectable levels of HO-1 (Fig. 3B and C, respectively). In marked contrast, the majority of monocytes from acute Kawasaki disease expressed low but significant levels of HO-1 (Fig. 3F). Furthermore, a small fraction of these cells were strongly positive for HO-1, as indicated by an arrow.
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). PBMC from acute Kawasaki disease (Fig. 4B, 1–5) and acute bacterial infection (Fig. 4B, 6–10) showed significant levels of HO-1 mRNA, but PBMCs from acute infectious mononucleosis (Fig. 4B, 11–14) did not express HO-1 mRNA.
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). In normal controls, PBMCs expressed low but detectable levels of HO-1 mRNA, although the actual levels of HO-1 mRNA expression varied significantly among individuals. HO-1/GAPDH ratio rarely exceeded 0.5 in normal controls. In acute Kawasaki disease, PBMCs expressed significantly higher levels of HO-1 mRNA than in normal controls. It is intriguing that a fraction of patients with Kawasaki disease expressed only low levels of HO-1 mRNA, comparable with normal controls. PBMCs from acute infectious mononucleosis expressed lower levels of HO-1 mRNA than controls. Both acute viral infection and bacterial infection were associated with significant levels of HO-1 mRNA expression by PBMCs. However, the levels of HO-1 mRNA expression varied significantly from 0.2 to 2.9.
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It’s been shown that HO-1 is preferentially expressed by specific types of cells in vivo (14, 19). Goda et al. reported that hepatic Kupffer cells express significant level of HO-1 without exogenous oxidative stress, whereas hepatic parenchymal cells produce HO-2 (19). It was strongly indicated by their study that HO-1 production by Kupffer cells serve as potent regulator of hepatic microcirculation. Spleen is the organ in which large level of HO activity is constantly detected (8). Splenic macrophages undergoing phagocytosis are actively producing HO-1 in normal spleen (unpublished observation). We also reported that renal tubular epithelial cells produced significant level of HO-1 regardless the nature of primary kidney diseases (14). The findings suggest that HO-1 is constantly produced by these cells in response to oxidative stress provided daily as glomerular filtrate. These characteristic distributions of HO-1-producing cells in vivo may reflect the frequency and intensity of oxidative stress encountered by the particular cells and may not directly reflect the ability of the selected cell types to produce HO-1 de novo. It is essential therefore, to stimulate different cells in vitro with various oxidative stresses and see if these cells have distinct patterns of HO-1 production, to understand the physiological significance of HO-1 in vivo.
The present study clearly indicates that monocytes are the principal source of HO-1 within PBMCs. The selective HO-1 production by monocytes is transcriptionally regulated, as HO-1 mRNA expression is restricted to monocytes after different types of stimulation. It’s been reported by Menzel et al. that peripheral blood lymphocytes are the source of HO-1 under oxidative stress and they suggested that the detection of HO-1 production by lymphocytes serve as a sensitive indicator of in vivo exposure to heavy metals such as arsenite or cadmium (20). They obtained PBMCs by Ficoll/Hypaque density gradient centrifugation and used this as lymphocytes. They also analyzed HO-1 production by immunoblotting using whole mononuclear cell population. Schipper et al. examined serum and lymphocyte HO-1 levels in Alzheimer dementia and found that lymphocyte HO-1 levels are significantly reduced in these patients as compared with age-matched controls. However, they also used PBMC separated from peripheral blood by Ficoll/Hypaque as lymphocytes (21). Based on our results, it is clear that HO-1 production by PBMCs depend largely on monocytes, but not on lymphocytes. Selective evaluation of monocyte HO-1 production by a flow cytometry, or immunohistochemical identification of HO-1 producing cells will certainly offer more sensitive and specific results to evaluate the significance of HO-1 production by peripheral blood leukocytes in various disease states.
Monocyte activation is frequently observed during acute inflammatory illnesses. Excessive and uncontrolled activation of monocyte/macrophage may lead to overproduction of proinflammatory cytokines and systemic organ dysfunction (3–5). Monocyte/macrophage is also playing key roles in the pathogenesis of coronary artery lesions (22, 23). Kawasaki disease is also known to have activated circulating monocytes during the acute stage of the illness (24, 25). These monocytes may have certain functional role in the pathogenesis of the coronary artery injuries. Collectively, activation of monocytes is the cardinal feature of systemic inflammation in various illnesses and anti-inflammatory therapeutic strategy is partly targeted at the regulation of the monocyte functions. In this regard, inhibition of one of the monocyte-derived proinflammatory cytokines, TNF, is becoming a promising treatment of recalcitrant rheumatic diseases (6, 7).
Recently, there is increasing evidence that HO-1 and its major metabolites, CO and bilirubin, act as potent anti-inflammatory mediators. There are several lines of evidences to support this notion. First, the abolition of HO-1 activity by gene targeting rendered the mice extremely susceptible to oxidative stress (26). Furthermore, our own experience with the first case of human HO-1 deficiency showed that persistent, systemic inflammation is the principal feature of the HO-1 defect (13). Second, the transfer of HO-1 gene reduced the level of inflammation and the risk of rejection of transplanted organs, suggesting that presence of excessive HO-1 reduce the level of inflammatory immune reactions (27). CO acts on monocytes to regulate its production of proinflammatory cytokines and enhancing IL-10 production (12). Third, polymorphism in HO-1 promoter region is closely associated with the risk of restenosis after angioplasty (28). The number of GT repeats seems to affect the expression of HO-1 mRNA in response to oxidative stresses (29). These findings indicate clearly that HO-1 and CO function as anti-inflammatory mediators in vivo.
Our findings show that monocyte HO-1 production is upregulated in vivo in various acute inflammatory illnesses. It is suggested from these data that HO-1 regulate the production of proinflammatory cytokines and protect the organs and cells from irreversible damages. Of particular importance is that monocyte HO-1 production is detectable even in normal control individuals, suggesting that circulating monocytes are constantly exposed to low, but significant levels of oxidative stress. It is intriguing that monocytes from acute Kawasaki disease are not always expressing high levels of HO-1 mRNA, but some of the cases are associated with very low HO-1 mRNA expression. We are currently investigating whether cases with low HO-1 production are related to significant coronary artery injury or whether it is associated with long GT repeats within HO-1 promotor region. Preliminary study indicates that circulating monocytes from Kawasaki disease are distinct from monocytes from other acute inflammatory diseases. Significant fractions of monocytes express low levels of surface HLA-DR and CD16, indicating that they are of amnestic, nonfunctioning monocytes. Therefore, the patterns of surface antigen expression and HO-1 production may be useful parameters of monocytes functions in vivo. Further studies are necessary to delineate the significance of monocyte HO-1 production in systemic inflammatory diseases.
Acknowledgments
Special thanks are given to Prof. Makoto Suematsu for providing the monoclonal antibody and for his critical comments. We appreciate technical assistance of H. Matsukawa and M. Kitakata. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan; a grant from the Ministry of Health, Labor and Welfare of Japan.
Footnotes
1 To whom requests for reprint should be addressed at Department of Laboratory Sciences, School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan. E-mail: yachie{at}med.kanazawa-u.ac.jp 
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