Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis.

We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs in the milieu of the inflamed joint of RA patients.     

Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis.

Cells within the synovial tissue may recruit mononuclear phagocytes into the synovial fluid and tissues of arthritic patients. We investigated the production of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1) using sera, synovial fluid, synovial tissue, as well as macrophages and fibroblasts isolated from synovial tissues from 80 arthritic patients. MCP-1 levels were significantly higher (P less than 0.05) in synovial fluid from RA patients (mean 25.5 +/- 8.1 ng/ml [SE]) compared to synovial fluid from osteoarthritis (OA) patients (0.92 +/- 0.08), or from patients with other arthritides (2.9 +/- 1.5). MCP-1 levels in RA sera (8.44 +/- 2.33) were significantly greater than MCP-1 in normal sera (0.16 +/- 0.06). The quantities of RA synovial fluid IL-8, which is chemotactic for neutrophils and lymphocytes, and MCP-1 were strongly positively correlated (P less than 0.05). To examine the cellular source of MCP-1, RA synovial tissue macrophages and fibroblasts were isolated. Synovial tissue fibroblasts did not express MCP-1 mRNA, but could be induced to produce MCP-1 by stimulation with either IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), or LPS. In contrast, unlike normal peripheral blood monocytes or alveolar macrophages, RA synovial tissue macrophages constitutively expressed MCP-1 mRNA and antigen. Immunohistochemical analysis of synovial tissue showed that a significantly greater percentage of RA macrophages (50 +/- 8%) as compared to either OA macrophages (5 +/- 2) or normal macrophages (1 +/- 0.3) reacted with anti-MCP-1 antibodies. In addition, the synovial lining layer reacted with MCP-1 in both RA and OA synovial tissues. In contrast, only a minority of synovial fibroblasts (18 +/- 8%) from RA synovium were positive for immunolocalization of MCP-1. These results suggest that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with RA and that synovial tissue macrophages are the dominant source of this cytokine.     

Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implications for HIV- associated neurological disease

Mononuclear phagocytes (monocytes, macrophages, and dendritic cells) play major roles in human immunodeficiency virus (HIV) persistence and disease pathogenesis. Macrophage antigen presentation and effector cell functions are impaired by HIV-1 infection. Abnormalities of macrophage effector cell function in bone marrow, lung, and brain likely result as a direct consequence of cellular activation and HIV replication. To further elucidate the extent of macrophage dysfunction in HIV-1 disease, a critical activation-specific regulatory molecule, nitric oxide (NO.), which may contribute to diverse pathology, was studied. Little, if any, NO. is produced by uninfected human monocytes. In contrast, infection with HIV-1 increases NO. production to modest, but significant levels (2-5 microM). Monocyte activation (with lipopolysaccharide, tumor necrosis factor alpha, or through interactions with astroglial cells) further enhances NO. production in HIV-infected cells, whereas its levels are diminished by interleukin 4. These results suggest a possible role for NO. in HIV-associated pathology where virus-infected macrophages are found. In support of this hypothesis, RNA encoding the inducible NO synthase (iNOS) was detected in postmortem brain tissue from one pediatric AIDS patient with advanced HIV encephalitis. Corresponding iNOS mRNA was not detected in brain tissue from five AIDS patients who died with less significant brain disease. These results demonstrate that HIV-1 can influence the expression of NOS in both cultured human monocytes and brain tissue. This newly described feature of HIV-macrophage interactions suggests previously unappreciated mechanisms of tissue pathology that result from productive viral replication.     

Cadherin-11 provides specific cellular adhesion between fibroblast-like synoviocytes

Cadherins are integral membrane proteins expressed in tissue-restricted patterns that mediate homophilic intercellular adhesion. During development, they orchestrate tissue morphogenesis and, in the adult, they determine tissue integrity and architecture. The synovial lining is a condensation of fibroblast-like synoviocytes (FLS) and macrophages one to three cells thick. These cells are embedded within the extracellular matrix, but the structure is neither an epithelium nor an endothelium. Previously, the basis for organization of the synovium into a tissue was unknown. Here, we cloned cadherin-11 from human rheumatoid arthritis (RA)-derived FLS. We developed L cell transfectants expressing cadherin-11, cadherin-11 fusion proteins, and anti-cadherin-11 mAb. Cadherin-11 was found to be expressed mainly in the synovial lining by immunohistologic staining of human synovium. FLS adhered to cadherin-11-Fc, and transfection of cadherin-11 conferred the formation of tissue-like sheets and lining-like structures upon fibroblasts in vitro. These findings support a key role for cadherin-11 in the specific adhesion of FLS and in synovial tissue organization and behavior in health and RA.     

Correlation of the topographical arrangement and the functional pattern of tissue-infiltrating macrophages in giant cell arteritis.

End organ ischemia, fragmentation of elastic membranes, and aneurysm formation in patients with giant cell vasculitis results from an inflammation destroying the mural layers of large and medium sized arteries. Although the inflammatory infiltrate extends through all layers of the affected blood vessel, the most pronounced changes involve the intima and the internal elastic lamina. Analysis of the functional profile of tissue infiltrating CD68+ cells demonstrates that different subsets of macrophages can be distinguished. TGFbeta1-expressing CD68+ cells coproduce IL-1beta and IL-6, are negative for inducible nitric oxide synthase (iNOS), and exhibit a strong preference for localization in the adventitia. The adventitial homing of TGFbeta1+ CD68+ cells places them in the vicinity of IFN-gamma secreting CD4+ T cells which also accumulate in the exterior layer of the artery. Conversely, iNOS expressing CD68+ cells are negative for TGFbeta and are almost exclusively found in the intimal layer of the inflamed artery. The intimal-medial junction is the preferred site for 72-kD collagenase expressing CD68+ cells. Thus, TGFbeta1-producing macrophages colocalize with activated CD4+ T cells and home to an area of inflammation which is distant from the site of tissue damage but critical in regulating cellular influx, suggesting that TGFbeta1 functions as a proinflammatory mediator in this disease. iNOS- and 72-kD collagenase-producing macrophages accumulate at the center of pathology implying a role of these products in tissue destruction. These data indicate that the microenvironment controls the topographical arrangement as well as the functional commitment of macrophages.     

Inhibition of extracellular signal-regulated kinase suppresses endotoxin-induced nitric oxide synthesis in mouse macrophages and in human colon epithelial cells

Macrophages produce large amounts of nitric oxide (NO) in response to proinflammatory cytokines and lipopolysaccharide (LPS) by expressing inducible isoform of NO synthase (iNOS). We examined the role of extracellular signal-regulated kinase p42/44(MAPK) (Erk1/2) in signal transduction pathways leading to induction of NO synthesis in response to LPS in J774 mouse macrophages and T-84 human colon epithelial cells. LPS activated Erk1/2 and induced iNOS and subsequent NO production. Erk1/2 activation was inhibited by PD 98059, a specific inhibitor of mitogen-activated protein kinase kinase (Mek) that is an upstream activator of Erk1/2. At corresponding concentrations PD 98059 reduced LPS-induced NO formation by 40 to 50% by inhibiting iNOS expression in J774 and T-84 cells. Inhibition of iNOS expression was not mediated by nuclear factor-kappaB because PD 98059 had no effect on nuclear factor-kappaB activity in J774 macrophages. In addition, PD 98059 reduced LPS-induced L-arginine transport into the cells as measured in J774 macrophages, whereas the availability of tetrahydrobiopterin was not a limiting factor in NO production after PD 98059. Our results indicate that Erk1/2 activation mediates up-regulation but is not essential for LPS-induced iNOS expression.     

Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritides.

In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05). Immunohistochemical analysis as well as in situ hybridization showed that inducible NO synthase was predominantly expressed in synovial lining cells, endothelial cells, chondrocytes, and to a lesser extent, in infiltrating mononuclear cells and synovial fibroblasts. The synovial lining cells and the infiltrating cells expressing inducible NO synthase were identified where CD14+ cells were located. Together with morphological features, this suggests that they are type A synoviocytes. NO production from freshly isolated synoviocytes and chondrocytes was up-regulated by in vitro stimulation with a combination of IL-TNF-beta, TNF-alpha, and LPS. In summary, the present results suggest that NO is produced primarily by CD14+ synoviocytes, chondrocytes, and endothelial cells in inflammatory joints of arthritides. NO production can be upregulated by cytokines present in inflamed joints. The increased NO production may thus contribute to the pathological features in inflammatory arthritides.     

Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major

Previous studies with inhibitors of inducible nitric oxide synthase (iNOS) suggested that high-output production of nitric oxide (NO) is an important antimicrobial effector pathway in vitro and in vivo. Here, we investigated the tissue expression of iNOS in mice after infection with Leishmania major. Immunohistochemical staining with an iNOS-specific antiserum revealed that in the cutaneous lesion and draining lymph nodes (LN) of clinically resistant mice (C57BL/6), iNOS protein is found earlier during infection and in significantly higher amounts than in the nonhealing BALB/c strain. Similar differences were seen on the mRNA level as quantitated by competitive polymerase chain reaction. Anti-CD4 treatment of BALB/c mice not only induced resistance to disease, but also restored the expression of iNOS in the tissue. In situ, few or no parasites were found in those regions of the skin lesion and the draining LN which were highly positive for iNOS. By double labeling experiments, macrophages were identified as iNOS expressing cells in vivo. In the lesions of BALB/c mice, cells staining positively for transforming growth factor beta (TGF-beta), a potent inhibitor of iNOS in vitro, were strikingly more prominent than in C57BL/6, whereas no such difference was found for interleukin 4 or interferon gamma (IFN-gamma). In vitro, production of NO was approximately threefold higher in C57BL/6 than in BALB/c macrophages after stimulation with IFN-gamma. We conclude that the pronounced expression of iNOS in resistant mice is an important mechanism for the elimination of Leishmania in vivo. The relative lack of iNOS in susceptible mice might be a consequence of macrophage deactivation by TGF-beta and reduced responsiveness to IFN-gamma.     

Nurse-like cells from bone marrow and synovium of patients with rheumatoid arthritis promote survival and enhance function of human B cells.

Thymic nurse cells are known to interact with T cells and play a role in their functional maturation. However, the role of nurse cells in B cell maturation and differentiation is less well established, especially at extralymphoid sites. To address this issue, nurse-like cell clones from bone marrow and synovial tissue of patients with RA (RA-NLC) were established and characterized. RA-NLC constitutively expressed CD29, CD49c, CD54 (ICAM-1), CD106 (VCAM-1), CD157 (BST-1), and class I MHC molecules, and secreted IL-6, IL-7, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Bone marrow-derived and synovial RA-NLC differed in that the former secreted IL-7 and expressed a greater density of CD157 constitutively and after stimulation with IFNgamma, whereas the latter secreted G-CSF and more IL-6. Stimulation of both bone marrow and synovial RA-NLC induced expression of CD40 and class II MHC, but not CD154 (CD40L) or CD35. RA-NLC rescued peripheral B cells from spontaneous apoptosis and promoted survival of B cells for > 4 wk. B cell survival was blocked by antibodies to CD106 or CD157. RA-NLC also increased Ig production from B cells. After long-term culture (4-6 wk) with RA-NLC, but not alone or with fibroblasts, outgrowth of B cells was observed. All B cell lines derived from these cultures had been transformed by EBV, although the RA-NLC themselves were not infected with EBV. Precursor frequency analysis indicated that approximately 1 in 12,500 peripheral B cells could give rise to these EBV-transformed B cell lines upon coculture with RA-NLC. These results indicate that RA-NLC from bone marrow and synovium have the capacity to rescue B cells from spontaneous apoptosis, facilitate Ig production, and promote the outgrowth of EBV-transformed B lymphoblastoid cells. These findings suggest that RA-NLC may play a role in the local and systemic hyperreactivity of B cells characteristic of rheumatoid arthritis.     

1-alpha,25-Dihydroxyvitamin D3 regulates inducible nitric oxide synthase messenger RNA expression and nitric oxide release in macrophage-like RAW 264.7 cells

The expression of inducible nitric oxide synthase (iNOS) expression and release of nitric oxide (NO) from macrophages are markedly increased in granulomatous infections. Activation of macrophages 1alpha-hydroxylase results in an increase of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, the significance of this increased production is not completely understood. In this study, we analyzed 1,25(OH)(2)D(3) and NO production in patients with tuberculosis infection and hypercalcemia and used lipopolysaccharide (LPS) to stimulate RAW 264.7 cells in an attempt to assess iNOS expression and gaseous NO production regulated by 1,25(OH)(2)D(3). Peroxynitrite (OONO(-)) production and lactate dehydrogenase activity were also examined. Without additional stimulation, peripheral-blood mononuclear cells (PBMCs) from patients with tuberculosis converted more 25-hydroxyvitamin D(3) to 1,25(OH)(2)D(3) than did those from normal controls. These PBMCs released less NO than did those from control subjects, at baseline and in the stimulated state. We found that 1,25(OH)(2)D(3) dose-dependently inhibited iNOS messenger RNA expression of the LPS-stimulated RAW 264.7 cells and also significantly reduced the gaseous NO release and OONO(-) production. Paralleling the 1,25(OH)(2)D(3)-induced inhibition of NO release were reductions in OONO(-) and LDH production. In conclusion, 1,25(OH)(2)D(3) inhibited iNOS expression and reduced NO production by LPS-stimulated macrophages in the range of physiological doses. Inhibition of the NO surge was coupled with a reduction in OONO(-) and LDH production. Increased 1,25(OH)(2)D(3) production and decreased release of NO from the PBMCs of patients with tuberculosis and hypercalcemia were also noted. We propose that 1,25(OH)(2)D(3) production by macrophages may protect themselves against oxidative injuries caused by the NO burst. In the case of tuberculosis infection, increased 1,25(OH)(2)D(3) synthesis may further contribute to the development of an unwanted phenomenon-hypercalcemia.     

Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase

Nitric oxide (NO) synthase (iNOS) is required for the resolution of acute cutaneous leishmaniasis in resistant C57BL/6 mice. As is the case in several other infections, the clinically cured host organism still harbors small amounts of live Leishmania major parasites. Here, we demonstrate lifelong expression of iNOS at the site of the original skin lesion and in the draining lymph node of long-term-infected C57BL/6 mice. iNOS activity in the lymph node was dependent on CD4+, but not on the CD8+ T cells. By double labeling techniques, iNOS and L. major were each found in macrophages (F4/80+, BM-8+, and/or MOMA-2+) and dendritic cells (NLDC-145+), but not in granulocytes or endothelial cells. In situ triple labeling of lymph node sections revealed that approximately 30-40% of the L. major foci were associated with iNOS- positive macrophages or dendritic cells. The majority of the L. major foci (60-70%), however, was located in areas that were negative for both iNOS and the macrophage and dendritic cell markers. In L. major- infected C57BL/6 mice, which had cured their cutaneous lesions, administration of L-N6-iminoethyl-lysine (L-NIL), a potent inhibitor of iNOS, led to a 10(4)-10(5)-fold increase of the parasite burden in the cutaneous and lymphoid tissue and caused clinical recrudescence of the disease. Persistent expression of iNOS and resumption of parasite replication after application of L-NIL was also observed in resistant C3H/HeN and CBA/J mice. We conclude that iNOS activity is crucial for the control of Leishmania persisting in immunocompetent hosts after resolution of the primary infection. Failure to maintain iNOS activity might be the mechanism underlying endogenous reactivation of latent infections with NO-sensitive microbes during phases of immunosuppression.     

Apoptosis in rheumatoid arthritis synovium.

RA synovial tissue (ST) was studied to determine if and where apoptosis occurs in situ. Genomic DNA was extracted from 5 RA and 1 osteoarthritis ST samples. Agarose gel electrophoresis demonstrated DNA ladders characteristic for apoptosis from each tissue. In situ and labeling (ISEL) was used to identify DNA strand breaks consistent with apoptosis in frozen sections. 12 RA and 4 osteoarthritis ST were studied by ISEL and all were positive, but only 2 of 4 normal tissues were positive. The primary location of apopotic cells was the synovial lining. Some sublining cells were also positive, but lymphoid aggregate staining was conspicuously absent. Immunohistochemistry and ISEL were combined and showed that the lining cells with DNA strand breaks were mainly macrophages, although some fibroblastlike cells were also labeled. Sublining cells with fragmented DNA included macrophages and fibroblasts, but T cells in lymphoid aggregates, which expressed large amounts of bcl-2, were spared. DNA strand breaks in cultured fibroblastlike synoviocytes was assessed using ISEL. Apoptosis could be induced by actinomycin D, anti-fas antibody, IL-1, and TNF-alpha but not by IFN-gamma. Fas expression was also detected on fibroblast-like synoviocytes using flow cytometry. Therefore, DNA strand breaks occur in synovium of patients with arthritis. Cytokines regulate this process, and the cytokine profile in RA (high IL-1/TNF; low IFN-gamma) along with local oxidant injury might favor induction of apoptosis.     

The altered tumoricidal capacity of macrophages isolated from tumor- bearing mice is related to reduce expression of the inducible nitric oxide synthase gene

Nitric oxide (NO) is a major effector molecule in the destruction of tumor cells by activated macrophages. However, in many cases, developing neoplasms appear to be capable of impairing steps in the complex process leading to NO production as a means of avoiding immune destruction. After activation with lipopolysaccharide (LPS), peritoneal- elicited macrophages (PEM) from mice bearing mammary tumors display alterations in their ability to lyse tumor cells due to reduced production of NO. In contrast, when these same cells are stimulated with LPS in combination with interferon gamma (IFN-gamma), they are able to produce NO and lyse targets at normal levels. Since tumor- associated macrophages are intimately associated with the cells of the developing tumor, their ability to produce NO and lyse tumor targets is likely to be more relevant to controlling tumor growth. This population of macrophages exhibited a more profound inability to produce NO and lyse targets and, unlike the PEM, was not able to upregulate these functions even when treated with combinations of LPS and IFN-gamma. Northern and Western blots revealed that inducible nitric oxide synthase (iNOS) mRNA and protein levels correlated directly with the ability of each macrophage population to produce NO, and the levels of these macromolecules were altered sufficiently in tumor bearers' macrophages to account for the diminished NO production described. These results indicate that a spatial gradient of suppression of macrophage cytolytic activity and iNOS expression exists in mammary tumor-bearing mice, whereby macrophages from within the tumor exhibit a more pronounced suppression than the more distally located PEM. This suppression may be due to proximity of the macrophages to the developing tumor, macrophage maturational state, or both.     

Functional profile of tissue-infiltrating and circulating CD68+ cells in giant cell arteritis. Evidence for two components of the disease.

Macrophages represent a critical component in the inflammatory lesions of giant cell arteritis. By combining immunohistochemistry and in situ hybridization, we have analyzed the functional heterogeneity of tissue-infiltrating macrophages in patients with untreated vasculitis. 20% of macrophages in temporal artery tissue synthesized IL-6-specific mRNA and produced IL-6 and IL-1 beta proteins. IL-6 and IL-1 beta production was not limited to CD68+ cells in the lymphoid aggregates but was a feature of CD68+ cells dispersed throughout the tissue. 50% of tissue-infiltrating CD68+ cells synthesized 72-kD type IV collagenase. Only a small subset of CD68+ cells produced cytokines as well as collagenase, indicating functional specialization or distinct differentiation stages of CD68+ cells in the inflamed tissue. Activation of CD68+ cells was not restricted to tissue-infiltrating cells. Expression of IL-6 and IL-1 beta was found in 60-80% of circulating monocytes of patients with untreated giant cell arteritis, whereas collagenase production was restricted to tissue macrophages. IL-6 and IL-1 beta production by the majority of circulating monocytes was a shared feature of patients with giant cell arteritis and polymyalgia rheumatica but was not found in rheumatoid arthritis. These data suggest that giant cell arteritis has two components of disease, an inflammatory reaction in vessel walls and a systemic activation of monocytes. Systemic monocyte activation can manifest independently without vasculitis as exemplified in patients with polymyalgia rheumatica.     

Regulation by endogenous interleukin-1 of mRNA expression of healing-related factors in gastric ulcers in rats.

We investigated the role of endogenous interleukin (IL)-1 in the mRNA expression of cyclooxygenase (COX)-1, COX-2, inducible nitric oxide synthase (iNOS), cytokine-induced neutrophil chemoattractant (CINC)-1, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and transforming growth factor (TGF)-beta1 in acetic acid-induced gastric ulcers in rats. IL-1beta mRNA was not detected in the normal or intact mucosa of ulcerated stomachs, but its expression was induced in the ulcerated tissue. IL-1beta immunoreactivity was observed in macrophages/monocytes and fibroblasts in the ulcer base. COX-2, iNOS, and CINC-1 mRNAs were expressed by ulceration. EGF, bFGF, HGF, and TGF-beta1 mRNA expression was detected in the normal mucosa, and their levels were significantly elevated by ulceration. In contrast, COX-1 mRNA level did not differ between the normal and ulcerated tissues. In a culture of isolated ulcer bases, block of IL-1 with IL-1 receptor antagonist (IL-1RA) dose-dependently and significantly reduced the mRNA levels of COX-2, iNOS, CINC-1, HGF, and bFGF. In contrast, COX-1, EGF, and TGF-beta1 mRNA expression was not affected by IL-1RA. IL-1RA dose-dependently reduced prostaglandin E(2) production, total and iNOS activities, neutrophil chemotactic activity, and growth-promoting activity toward gastric epithelial cells in the ulcer base. Finally, the administration of IL-1RA caused a significant impairment of ulcer healing. These results indicate that IL-1, expressed in macrophages/monocytes and fibroblasts in the ulcer base, might up-regulate the mRNA expression of COX-2, iNOS, CINC-1, HGF, and bFGF, thereby contributing to gastric ulcer healing in rats.