Mechanisms for monocyte activation in co-culture with autologous tumor spheroids

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro secrete interleukin (IL)-6 upon 24h in co-culture. Presently, the aim was to study the mechanisms of this monocyte secretion. Paraformaldehyde (0.1% for 2min) or actinomycin-D (1 microg/ml for 24h) pre-treatment of the F-spheroids abolished the monocyte IL-6 co-culture response. Addition of glucose (100mM) or mannose (100mM), and to some extent galactose (100mM), but not fructose (100mM) to the co-cultures, partly inhibited the monocyte IL-6 co-culture response, but such addition did not inhibit the in vitro monocyte lipopolysaccharide (LPS)-generated IL-6 secretion. When mannose was added to the co-cultures, monocyte IL-6 mRNA expression was eradicated in malignant co-cultures and reduced to a low level in benign co-cultures. Addition of mouse anti-human beta(1)-integrin (anti-CD29) antibody (2 microg/ml) diminished the IL-6 co-culture response but not the monocyte LPS-generated IL-6 response. In conclusion, the monocyte IL-6 co-culture response is dependent on live spheroids and to some extent on direct contact with the F-spheroids, possibly via lectin-like receptor(s), the mannose receptor and beta(1)-integrin.     

Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-alpha, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-alpha production.     

Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte–macrophage interaction

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis.Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1.In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.     

LILRA2 selectively modulates LPS-mediated cytokine production and inhibits phagocytosis by monocytes

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.     

Increased monocyte MCP-1 production in acute alcoholic hepatitis.

Monocyte chemoattractant protein-1 (MCP-1) is a potent mononuclear cell-specific chemotactic protein. MCP-1 is a candidate chemoattractant for activation and hepatic infiltration of mononuclear cells in alcoholic hepatitis (AH). Blood was collected from 15 patients with AH (mean bilirubin 17.6+/-3.5 mg/dl; normal 0. 2-1.0 mg/dl) on admission and at time points for up to 6 months. Peripheral blood monocytes were isolated and MCP-1 production assessed by measuring MCP-1 concentrations in monocyte culture supernatants after overnight (20 h) incubation. Monocytes from normal subjects did not product detectable MCP-1 unless stimulated with endotoxin (LPS;5 microg/ml). The mean level of constitutive MCP-1 from AH patient monocytes was 4694+/-2432 pg/ml 20 h on admission. The mean MCP-1 level for LPS-treated monocytes was 4903+/-1540 pg/ml 20 h for normal subjects and was significantly elevated in AH patients to 11589+/-3266 pg/ml/20 h. AH patient monocyte MCP-1 production was decreased in vitro when monocytes were treated with N-acetylcysteine (5 mM) and also decreased over the 6-month study as the patients improved clinically. MCP-1 plasma levels were below the detection limits of the assay used in both AH patients and normal subjects. Thus, monocytes from AH patients not only constitutively product MCP-1, but also produce higher levels of MCP-1 with endotoxin stimulation. Further studies are needed to clarify the role of MCP-1 in the activation and hepatic infiltration of mononuclear cells in alcoholic liver disease.     

Regulatory role of thioredoxin in homocysteine-induced monocyte chemoattractant protein-1 secretion in monocytes/macrophages

We have previously shown that homocysteine (Hcy) can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes. Here, we show that Hcy upregulates expression of an important antioxidative protein, thioredoxin (Trx), via NADPH oxidase in human monocytes in vitro. The increase of Trx expression and activity inhibited Hcy-induced ROS production and MCP-1 secretion. Of note, 2-week hyperhomocysteinemia (HHcy) ApoE^−/− mice showed accelerated lesion formation and parallel lower Trx expression in macrophages than ApoE^−/− mice, suggesting that HHcy-induced sustained oxidative stress in vivo might account for impaired Trx and hence increased ROS production and MCP-1 secretion from macrophages, and subsequently accelerated atherogenesis.     

Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells

Many acute and chronic lung diseases are characterized by the presence of increased numbers of activated macrophages. These macrophages are derived predominantly from newly recruited peripheral blood monocytes and may play a role in the amplification and perpetuation of an initial lung insult. The process of inflammatory cell recruitment is poorly understood, although the expression of inflammatory cell-specific chemoattractants and subsequent generation of chemotactic gradients is likely involved. Although immune cells such as macrophages and lymphocytes are known to generate several inflammatory cell chemoattractants, parenchymal cells can also synthesize and secrete a number of bioactive factors. We now demonstrate the generation of significant monocyte chemotactic activity from tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta-treated pulmonary type II-like epithelial cells (A549). The predominant inducible monocyte chemotaxin had an estimated molecular mass of approximately 14-15 kDa and was neutralized by specific antibody to human monocyte chemotactic protein-1 (MCP-1). Induction of activity was accompanied by increases in steady-state mRNA level for MCP-1. These data are consistent with the induction of MCP-1 expression from A549 cells by TNF and IL-1. MCP-1 production from A549 cells could be induced by lipopolysaccharide (LPS)-stimulated alveolar macrophage (AM)-conditioned media, but not by LPS alone. The inducing activity in AM-conditioned media was neutralized with specific antibodies to IL-1 beta, but not TNF-alpha. Our findings suggest that the alveolar epithelium can participate in inflammatory cell recruitment via the production of MCP-1 and that cytokine networking between contiguous alveolar macrophages and the pulmonary epithelium may be essential for parenchymal cell MCP-1 expression.     

Obesity-associated mouse adipose stem cell secretion of monocyte chemotactic protein-1

Studies showed that monocyte chemotactic protein-1 (MCP-1) concentrations are increased in obesity. In our current study, we demonstrate that plasma MCP-1 level in leptin-deficient ob/ob mice is significantly higher than in lean mice. Furthermore, we determined that basal adipose tissue MCP-1 mRNA levels are significantly higher in ob/ob mice compared with lean mice. To determine the mechanisms underlying obesity-associated increases in plasma and adipose tissue MCP-1 levels, we determined adipose tissue cell type sources of MCP-1 production. Our data show that adipose tissue stem cells (CD34(+)), macrophages (F4/80(+)), and stromal vascular fraction (SVF) cells express significantly higher levels of MCP-1 compared with adipocytes under both basal and lipopolysaccharide (LPS)-stimulated conditions. Furthermore, basal and LPS-induced MCP-1 secretion levels were the same for both adipose F4/80(+) and CD34(+) cells, whereas adipose CD34(+) cells have twofold higher cell numbers (30% of total SVF cells) compared with F4/80(+) macrophages (15%). Our data also show that CD34(+) cells from visceral adipose tissue depots secrete significantly higher levels of MCP-1 ex vivo when compared with CD34(+) cells from subcutaneous adipose tissue depots. Taken together, our data suggest that adipose CD34(+) stem cells may play an important role in obesity-associated increases in plasma MCP-1 levels.     

Role of redox factor-1 in hyperhomocysteinemia-accelerated atherosclerosis

Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. We have previously shown that homocysteine can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes in vitro. In the present study, we investigated whether redox factor-1 (Ref-1) is involved in HHcy-accelerated atherosclerosis. We used a mild HHcy animal model, aortic roots and peritoneal macrophages were isolated for immunohistochemistry and Western blotting, from apoE-/- and C57BL/6J mice fed a high Hcy diet (1.8 g/L) for 4 or 12 weeks. Four-week HHcy apoE-/- mice showed more plaques and significantly increased immunostaining of Ref-1 and MCP-1 in foam cells, and HHcy mice showed enhanced Ref-1 expression in peritoneal macrophages. To explore the mediating mechanism, incubation with Hcy (100 microM) increased Ref-1 protein level and translocation in human monocytes in vitro. In addition, Hcy-induced NADPH oxidase activity mediated the upregulation of Ref-1. Furthermore, overexpressed Ref-1 upregulated NF-kappaB and MCP-1 promoter activity, and antisense Ref-1 reduced Hcy-induced NF-kappaB DNA-binding activity and MCP-1 secretion. These data indicate that Hcy-induced ROS upregulate the expression and translocation of Ref-1 via NADPH oxidase, and then Ref-1 increases NF-kappaB activity and MCP-1 secretion in human monocytes/macrophages, which may accelerate the development of atherosclerosis.     

Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine,MCP-1 and activation of TLR4 and Gi/o proteins signaling

The cytokine resistin and the chemokine fractalkine (FKN) were found at increased levels in human atherosclerotic plaque, in the subendothelium, but their role in this location still needs to be characterized. Recently, high local resistin in the arterial vessel wall was shown to contribute to an enhanced accumulation of macrophages by mechanisms that need to be clarified. Our recent data showed that resistin activated smooth muscle cells (SMC) by up-regulating FKN and MCP-1 expression and monocyte chemotaxis by activating toll-like receptor 4 (TLR4) and Gi/o proteins. Since in the vessel wall both endothelial cells (EC) and SMC respond to cytokines and promote atherosclerosis, we questioned whether subendothelial resistin (sR) has a role in vascular cells cross-talk leading to enhanced monocyte transmigration and we investigated the mechanisms involved. To this purpose we used an in vitro system of co-cultured SMC and EC activated by sR and we analyzed monocyte transmigration. Our results indicated that: (1) sR enhanced monocyte transmigration in EC/SMC system compared to EC cultured alone; (2) sR activated TLR4 and Gi/o signaling in EC/SMC system and induced the secretion of more FKN and MCP-1 compared to EC cultured alone and used both chemokines to specifically recruit monocytes by CX3CR1 and CCR2 receptors. Moreover, FKN produced by resistin in EC/SMC system, by acting on CX3CR1 on EC/SMC specifically contributes to MCP-1 secretion in the system and to the enhanced monocyte transmigration. Our study indicates new possible targets for therapy to reduce resistin-dependent enhanced macrophage infiltration in the atherosclerotic arterial wall.     

“Spexin improves adipose tissue inflammation and macrophage recruitment in obese mice”

Spexin (SPX) is a novel adipokine related to many metabolic effects, such as gastrointestinal movements, insulin and glucose homeostasis, lipid metabolism and energy balance. This study evaluates the role of SPX in the improvement of the metabolic and inflammatory profile in fructose-rich-diet obese mice. Adult Swiss mice were supplemented or not with fructose (20% in tap water, FRD and CTR, respectively) for 10 weeks. The last ten days, mice were treated or not with SPX (ip. 29 μg/Kg/day, FRD-SPX and CTR-SPX, respectively). A positive correlation was observed between body weight prior to treatment and weight loss after SPX challenge. Moreover, plasma and liver triglycerides and adipose tissue (AT) features (mass, adipocyte hypertrophy, mRNA of leptin) were improved. SPX also induced a reduction in epididymal AT (EAT) expression of TNFα, IL1β and IL6 and an improvement in IL10 and CD206. M1 macrophages in EAT, principally the Ly6C⁻ populations (M1a and M1b), were decreased. Adipocytes from FRD-SPX mice induced less macrophage activation (IL6, mRNA and secretion) than FRD after overnight co-culture with the monocyte cell line (RAW264.7) in stimulated conditions (M1 activation, LPS 100 ng/mL). Finally, in vitro, monocytes pre-incubated with SPX and stimulated with LPS showed decreased inflammatory mRNA markers compared to monocytes with LPS alone. In conclusion, SPX decreased body weight and improved the metabolic profile and adipocyte hypertrophy. Inflammatory Ly6C⁻ macrophages decreased, together with inflammatory marker expression. In vitro studies demonstrate that SPX induced a decrease in M1 macrophage polarization directly or through mature adipocytes.     

Tolerogenic probiotics Lactobacillus delbrueckii and Lactobacillus rhamnosus promote anti-inflammatory profile of macrophages-derived monocytes of newly diagnosed patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is known as an autoimmune disorder that is characterized by the breakdown of self-tolerance, resulting in disease onset and progression. Macrophages have been implicated as a factor in the development of SLE through faulty phagocytosis of dead cells or an imbalanced M1/M2 ratio. The study aimed to investigate the immunomodulatory effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on M1 and M2 macrophages in new case lupus patients. For this purpose, blood monocytes were collected from lupus patients and healthy people and were cultured for 5 days to produce macrophages. For 48 h, the macrophages were then cocultured with either probiotics or lipopolysaccharides (LPS). Flow cytometry and real-time polymerase chain reaction were then used to analyze the expression of cluster of differentiation (CD) 14, CD80, and human leukocyte antigen – DR (HLADR) markers, as well as cytokine expression (interleukin [IL]1-β, IL-12, tumor necrosis factor α [TNF-α], IL-10, and transforming growth factor beta [TGF-β]). The results indicated three distinct macrophage populations, M0, M1, and M2. In both control and patient-derived macrophage-derived monocytes (MDMs), the probiotic groups showed a decrease in CD14, CD80, and HLADR expression compared to the LPS group. This decrease was particularly evident in M0 and M2 macrophages from lupus patients and M1 macrophages from healthy subjects. In addition, the probiotic groups showed increased levels of IL-10 and TGF-β and decreased levels of IL-12, IL1-β, and TNF-α in MDMs from both healthy and lupus subjects compared to the LPS groups. Although there was a higher expression of pro-inflammatory cytokines in lupus patients, there was a higher expression of anti-inflammatory cytokines in healthy subjects. In general, L. delbrueckii and L. rhamnosus could induce anti-inflammatory effects on MDMs from both healthy and lupus subjects.     

Activation of PKC-beta isoforms mediates HNE-induced MCP-1 release by macrophages

4-Hydroxynonenal (HNE) in the concentration range detectable in many pathophysiologic conditions is able to modulate signal transduction cascades and gene expression. Here, we report the stimulating effect of 1 microM HNE on the release of the monocyte chemotactic protein-1 (MCP-1) by murine macrophages. MCP-1-increased export following 1-h cell treatment with HNE proved to be comparable to that exerted by standard amounts of bacterial lipopolysaccharide (LPS). However, the key molecular event in HNE-induced secretion of MCP-1 appeared to be the increased activity of beta-PKC isoforms, which are recognized as playing a role in the regulation of cell protein transport and secretion. On the other hand, in LPS-stimulated cells, the delta isoform was seen to be involved and was probably related to LPS-mediated effects on MCP-1 expression and synthesis. In conclusion, HNE might interact with other pro-inflammatory stimuli, like LPS, in a concerted amplification of MCP-1 production and secretion.     

Inhibition of macrophage priming by sulfatide from Mycobacterium tuberculosis

Sulfatide from the outer surface of Mycobacterium tuberculosis blocked priming in cultured human monocytes. Monocytes were primed in vitro with either lipopolysaccharide (LPS) or interferon-gamma. Primed monocytes released increased amounts of superoxide anion (O2-) when stimulated with formyl-methionyl-leucyl-phenylalanine or with phorbol myristate acetate. Primed monocytes also showed increased phagocytosis of sheep erythrocytes and increased release of interleukin 1. When primed monocytes were treated with 10 micrograms/ml of sulfatide, these enhanced functions, characteristic of primed monocytes, returned to levels found in unprimed monocytes. (With respect to these functions and others, monocytes or macrophages primed in vitro by exposure to LPS or interferon-gamma resemble macrophages activated in vivo by infection. In vivo, activated macrophages provide non-specific resistance to infection). Inhibition of priming by sulfatide could be detected within 10 min, but maximum effect of sulfatide required 3 to 5 hr. Sulfatide had no effect on O2- release, if it was added after the cells had been stimulated by PMA, suggesting that sulfatide did not inhibit enzymes involved in formation of O2-, but rather that sulfatide inhibited priming. Increasing the amounts of LPS or interferon-gamma did not counteract the effects of sulfatide. Sulfatide did cause monocytes to release some prostaglandin E2 (less than 1 nM), but the amount was not sufficient to inhibit monocyte functions. The effect of sulfatide was not blocked by indomethacin. Other sulfated compounds and other products of mycobacteria did not produce the sulfatide effect. We conclude that M. tuberculosis has on its outer surface a chemical that directly interferes with monocyte priming. In vivo, M. tuberculosis might use sulfatide to block macrophage activation and thereby resist being killed by macrophages.     

Lipopolysaccharide induces actin reorganization and tyrosine phosphorylation of Pyk2 and paxillin in monocytes and macrophages

The bacterial endotoxin LPS is a potent stimulator of monocyte and macrophage activation and induces adhesion of monocytes. Morphological changes in response to LPS have not been characterized in detail, however, nor have the signaling pathways mediating LPS-induced adhesion been elucidated. We have found that LPS rapidly induced adhesion and spreading of peripheral blood monocytes, and that this was inhibited by the Src family kinase inhibitor PP1 and the phosphatidylinositide 3-kinase inhibitor LY294002. LPS also stimulated actin reorganization, leading to the formation of filopodia, lamellipodia, and membrane ruffles in Bac1 mouse macrophages. Proline-rich tyrosine kinase 2 (Pyk2), a tyrosine kinase related to focal adhesion kinase, and paxillin, a cytoskeletal protein that interacts with Pyk2, were both tyrosine phosphorylated in response to LPS in monocytes and macrophages. Both tyrosine phosphorylation events were inhibited by PP1 and LY294002. Adhesion also stimulated tyrosine phosphorylation of Pyk2 and paxillin in monocytes, and this was further enhanced by LPS. Finally, Pyk2 and paxillin colocalized within membrane ruffles in LPS-stimulated cells. These results indicate that LPS stimulation of monocytes and macrophages results in rapid morphological changes and suggest that Pyk2 and/or paxillin play a role in this response.     

Monocyte chemoattractant protein-1 induces scavenger receptor expression and monocyte differentiation into foam cells

Accumulation of monocytes and the entrapment of oxidized-low-density lipoprotein (ox-LDL) in monocytes are important in the differentiation into "foam" macrophages and the pathogenesis of atherosclerosis. We investigated the role of monocyte chemoattractant protein-1 (MCP-1) in the expression of scavenger receptor (SCR) by using resting monocytes prepared by counterflow centrifugal elutriation. Our results showed that: (1) MCP-1 increased the expression of CD36 SCR by flow cytometric analysis. (2) MCP-1 increased incorporation of 125I-labeled ox-LDL and oil red O staining. (3) MCP-1 and ox-LDL enhanced in vitro transendothelial monocyte migration. (4) These functions were mediated at least in part via extracellular signal-regulated kinase (ERK) pathway. (5) MCP-1 and ox-LDL did not induce monocyte proliferation. Our results imply that MCP-1 is involved in the inflammatory process of atherosclerosis through the induction of SCR expression via the ERK pathway and differentiation of monocytes into foam macrophages, as well as induction of monocyte migration.     

Functional IL-2 receptor beta (CD122) and gamma (CD132) chains are expressed by fibroblast-like synoviocytes: activation by IL-2 stimulates monocyte chemoattractant protein-1 production

The expression of the IL-2R alpha-, beta-, and gamma-chains, CD25, CD122, and CD132, respectively, was investigated on fibroblast-like synoviocytes (FLS) and dermal fibroblasts (DF). Both protein and mRNA for CD122 and CD132 were observed but there was no evidence of CD25 expression. Quantification of the Ag binding sites for CD122 showed that FLS expressed 4 times more receptor molecules than DF. The functional capability of these receptors was confirmed by the production of monocyte chemoattractant protein-1 (MCP-1) in direct response to stimulation by IL-2, which could be inhibited by neutralizing anti-CD122 mAb. Both rheumatoid arthritis (RA) and osteoarthritis (OA) FLS and DF spontaneously produced MCP-1 in culture over a similar range of concentrations. However, RA and OA FLS produced significantly greater levels of MCP-1 following stimulation by IL-2 and IL-1 beta; RA FLS produced significantly more MCP-1 than OA FLS. Addition of exogenous IL-2 caused a slight, but significant, decrease in MCP-1 production by DF. The addition of neutralizing anti-CD122 mAb to FLS cultures partially, but significantly, reduced the IL-2-induced MCP-1 secretion, but did not effect either the spontaneous or IL-1 beta-induced secretion of MCP-1. Increased tyrosine phosphorylation was observed in FLS lysates following 30-min incubation with IL-2. In conclusion, in the inflamed synovium, as activated T cells migrate through the sublining and lining layer, T cell-derived IL-2 may activate FLS to secrete MCP-1, thus recruiting macrophages into the rheumatoid synovium and perpetuating inflammation.