Monocyte-conditioned Medium Research Articles

Expression and a role of functionally coupled P2Y receptors in human dendritic cells

We investigated the physiology and function of P2Y receptors expressed in human dendritic cells (DCs) differentiated in vitro from CD14 + cells (DC-14). These were obtained after a 10 day stimulation period in GM-CSF, IL-4 and monocyte conditioned medium. DC-14 were found to express high amounts of MHC class II, B7, CD40 as well as CD83. The functional analysis, using single cell Ca 2+ imaging, demonstrated the expression of at least three subtypes of P2Y receptors. We further found using patch-clamp measurements that ATP evoked a pertussis toxin insensitive non-selective cation current with a peak current amplitude of −276±43 pA (holding potential −80 mV, n=23). This current was not Ca 2+-activated, since it was still observed under conditions of high intracellular Ca 2+ buffering and could be blocked by Gd 3+ (0.5 mM). In addition, intracellular application of GTP-γ-S (0.3 mM) also activated the current. Interestingly, DC-14 redirected the orientation of their dendrites as well as cell shape towards a pipette containing ATP as observed with time lapse microscopy. These data suggest that in human DCs, ATP acts via P2Y receptors and induces chemokine effects.

Interleukin 1beta mediates the modulatory effects of monocytes on LNCaP human prostate cancer cells.

Proliferative and secretory responses in androgen-sensitive prostate cancer LNCaP cells are regulated by steroid and peptide hormones and by differentiation-promoting substances. In the present study, we evaluated whether peripheral blood monocytes that exhibit anti-tumour activity in haematopoietic and solid tumours influence growth and secretion in the LNCaP cell line. For this purpose, LNCaP cells were incubated with monocyte-conditioned medium (MCM), and proliferation as well as expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) were assessed. Conditioned medium from monocytes reduced proliferation in a dose-dependent manner. Incubation with 40% MCM caused a 50% reduction in cell proliferation. AR protein decreased by 70% and PSA levels in supernatants from LNCaP cells were reduced by approximately 80% following treatment with MCM. We focused on the contribution of two major products of activated monocytes, prostaglandin E2 and interleukin 1beta (IL-1beta), to the MCM modulatory action. LNCaP cells treated with prostaglandin E2 showed neither a reduction in proliferation nor a down-regulation of AR and PSA levels. The effects of MCM on cellular proliferation, AR protein and PSA secretion were abolished by pretreatment of MCM with a neutralizing anti-IL-1beta antibody. In addition, recombinant IL-1beta was able to replace MCM for the inhibition of proliferation and down-regulation of AR and PSA proteins. LNCaP cells were shown to express the IL-1beta receptor type 1, which transduces IL-1beta signal. Our findings reveal that monocyte-derived IL-1beta inhibits the proliferation of androgen-responsive prostate tumour cells and reduces AR and PSA levels.

Cytokine-mediated Down-regulation of CYP1A1 in Hepa1 Cells

The activation of host defense mechanisms down-regulates microsomal cytochrome P450 in cell culture, humans, and animals. Investigation into various aspects of this effect using in vivo models has yet to define clearly the role that cytokines play in this phenomenon. The mechanism of down-regulation by immunostimulants, such as lipopolysaccharide (LPS), is explored with an in vitro model, utilizing a murine hepatoma (Hepa1) and a murine macrophage (IC-21) cell line. It is hypothesized that down-regulation of P450 activity by immunostimulants involves the activation of immune cells and the subsequent release of cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The effects of immunostimulation on P450 activity are assessed by ethoxyresorufin O-dealkylase, an assay that measures CYP1A activity in Hepa1 cells. Initial studies demonstrated that LPS added directly to hepatoma cells had no effect on the levels of CYP1A1 activity. In contrast, a significant down-regulation in CYP1A1 activity occurred when hepatoma cells were incubated with monocyte conditioned medium obtained by incubating LPS with IC-21 cells. When pentoxifylline, a TNF-α synthesis inhibitor, was co-administered with LPS to macrophages, the down-regulation of CYP1A1 activity was prevented. The direct administration of murine recombinant TNF-α to hepatoma cells resulted in a down-regulation of CYP1A1 activity. These results implicated the release of TNF-α from macrophages as an important step in the down-regulation of CYP1A1 by LPS.

Immature dendritic cells selectively replicate macrophagetropic (M-tropic) human immunodeficiency virus type 1, while mature cells efficiently transmit both M- and T-tropic virus to T cells.

Dendritic cells (DCs) can develop from CD14+ peripheral blood monocytes cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). By 6 days in culture, the cells have the characteristics of immature DCs and can be further induced to mature by inflammatory stimuli or by monocyte-conditioned medium. After infection with macrophagetropic (M-tropic) human immunodeficiency virus type 1 (HIV-1), monocytes and mature DCs show a block in reverse transcription and only form early transcripts that can be amplified with primers for the R/U5 region. In contrast, immature DCs cultured for 6 or 11 days in GM-CSF and IL-4 complete reverse transcription and show a strong signal when LTR/gag primers are used. Blood monocytes and mature DCs do not replicate HIV-1, whereas immature DCs can be productively infected, but only with M-tropic HIV-1. The virus produced by immature DCs readily infects activated T cells. Although mature DCs do not produce virus, these cells transmit both M- and T-tropic virus to T cells. In the cocultures, both DCs and T cells must express functional chemokine coreceptors for viral replication to occur. Therefore, the developmental stage of DCs can influence the interaction of these cells with HIV-1 and influence the extent to which M-tropic and T-tropic virus can replicate.

Mature Dendritic Cells Respond to SDF-1, but not to Several β-Chemokines

Immature dendritic cells (DCs) are highly motile, but after differentiation they stop migration. Chemokines are chemotactic cytokines that direct leukocyte trafficking, therefore we looked for the expression and function of chemokine receptors in immature and mature DCs. As a model, we used the human DCs that develop from CD14+ peripheral blood monocytes cultured with GM-CSF and IL-4. After 6-7 days in culture, these cells have the characteristics of immature DCs, but can be induced to mature further by inflammatory stimuli or by monocyte conditioned medium (MCM). Immature DCs express mRNA for CXCR4, CCR3 and CCR5. The receptors are expressed on the cell surface, as assessed with monoclonal antibodies, and are functional (with the exception of CCR3) as assessed by CA++ mobilization in response to specific chemokines. Further differentiation and maturation of DC in MCM causes a downregulation of expression and function of the beta-chemokine receptors, while CXCR4 still remains, and signals a calcium flux on mature DCs. We argue that the downregulation of beta-chemokine receptors during maturation helps to stop DC movement after T cells have been identified in lymphoid organs or at sites of delayed-type hypersensitivity.

Platelet-Derived Growth Factor and Pentoxifylline Modulation of Collagen Synthesis in Myofibroblasts

Fibroblast proliferation and extracellular matrix accumulation are two major events occurring in fibrosis. Hepatic stellate cells are the major collagen-producing cells of the liver and are transformed into proliferative myofibroblasts following activation. Whether proliferation and extracellular matrix production are regulated by the same cytokines is not known. Monocyte-conditioned medium obtained from pigs with yellow phosphorus-induced hepatic fibrosis increased the collagen production by cultured porcine myofibroblasts. Liver biopsies from these same fibrotic animals had increased levels of collagen α1(I) and α1(III) mRNA compared to control animals. Preincubation with platelet-derived growth factor (PDGF) B/B antibody significantly reduced the collagen-stimulating ability of the monocyte-conditioned medium. Recombinant PDGF stimulated proliferation in nonconfluent myofibroblasts and stimulated collagen production in confluent cultures of myofibroblasts without increasing cell number, suggesting that these events can occur independent of each other. Pentoxifylline and one of its active metabolites (metabolite-1) inhibited PDGF-stimulated collagen production in cultured porcine myofibroblasts. These results demonstrate the importance of PDGF in the pathogenesis of liver fibrosis and provide evidence that pentoxifylline interferes with PDGF-mediated events during experimental liver fibrosis.

Expression of Maturation-/Migration-Related Molecules on Human Dendritic Cells from Blood and Skin

Progress in dendritic cell research has been overwhelming in the past few years. This was made possible by the recent development of simple methods to generate large numbers of dendritic cells. These methods use as starting populations for culture either CD34 + progenitor cells from cord blood or bone marrow, or monocytes from peripheral blood. The latter approach is critically dependent on the combination of GM-CSF and interleukin 4. Such “priming cultures” yield populations of immature dendritic cells (CD83 −/CD86 ±/CD115 +/antigen uptake high/antigen processing high/T cell sensitization low). In order to generate mature dendritic cells a subsequent “differentiation culture” has to be added whereby monocyte-conditioned medium appears to be the optimal stimulus for maturation. This results in terminally mature dendritic cells (CD83 +/CD86 ++/CD115 −/antigen uptake low/antigen processing low/T cell sensitization high). We investigated the expression of some molecules involved in maturation and migration on human monocyte-derived dendritic cells from blood in comparison with dermal dendritic cells and epidermal Langerhans cells. We present a method to highly enrich epidermal Langerhans cells. Survival of purified Langerhans cells in culture is dependent on the presence of GM-CSF and TNF-α. During maturation a substantial part of the Langerhans cells loses expression of the cutaneous lymphocyte antigen (CLA); mature dendritic cells from the dermis are completely devoid of CLA. Similarly, CLA as well as CD15s (Sialyl Lewis x) and CD31 (PECAM-1) that can be readily detected on immature monocyte-derived dendritic cells are down-regulated upon maturation. CD68 expression is very low in cutaneous dendritic cells; in monocyte-derived dendritic cells this molecule is abundantly present. Subsets of monocyte-derived dendritic cells express E-cadherin; CD87 (urokinase plasminogen activator receptor) is weakly expressed on both immature and mature monocyte-derived dendritic cells. Taken together, these data suggest that the phenotype of monocyte-derived dendritic cells (E-cadherin low to negative, CD68 ++) is not indicative for a cutaneous destiny. Furthermore, the downregulation upon maturation of molecules involved in migration through vessel walls (CD31, CLA, CD15s) indicates that the entry of mature dendritic cells into lymphatic vessels may not be as rigidly regulated by adhesion molecules as the process of extravasation from blood vessels.

Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions.

Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-alpha/IL-1/IL-6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)-gamma and no IL-4 or IL-10. Production of IFN-gamma was significantly induced by addition of PGE2, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN-gamma(low), IL-4(neg) cells, TNF-alpha/IL-1/IL-6 promoted growth of IFN-gamma(intermediate), IL-4(neg) CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN-gamma production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-alpha/IL-1/IL-6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF-alpha/IL-1, IL-6 + PGE2-cultured DC seem to be optimal for generation of IFN-gamma-producing CD4/CD8+ T cells.

A Monocyte Conditioned Medium Is More Effective Than Defined Cytokines in Mediating the Terminal Maturation of Human Dendritic Cells

AbstractMature human dendritic cells can be generated in substantial numbers from nonproliferating progenitors in human blood using a two-step protocol. T cell–depleted mononuclear cells are first cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) and then exposed to monocyte conditioned medium (MCM). The dendritic cells generated using this approach are rendered terminally mature and are the most potent antigen presenting cells identified to date in humans. We sought to characterize factors in MCM that induce the terminal differentiation of dendritic cells. MCM contained substantial, although varying, quantities of several factors including tumor necrosis factor-α, IL-1β, IL-6, and interferon-α. However, none of the four factors, individually or in various combinations, could fully substitute for the MCM to generate irreversibly differentiated dendritic cells. The yields, percentage of cells expressing the mature phase marker CD83, and mixed leukocyte reaction–stimulatory function were lower when defined cytokines were used in the place of MCM. Therefore, the full maturation of dendritic cells, because it entails changes in many known cell and molecular properties, requires a number of different cytokines that are released in tandem from appropriately stimulated monocytes. We propose that MCM-matured dendritic cells will be the most effective adjuvants for immunotherapy in vivo.

Dendritic cells generated from blood precursors of chronic myelogenous leukemia patients carry the Philadelphia translocation and can induce a CML-specific primary cytotoxic T-cell response.

Dendritic cells (DC) are professional antigen-presenting cells specialized in the initiation of primary immune responses. We were interested to know whether mature DC can be grown in vitro from peripheral blood mononuclear cells (PBMC) of patients with chronic myelogenous leukemia (CML), and whether they carry the Philadelphia (Ph) translocation. Using a method recently described, DC were generated from PBMC precursors of 12 patients with CML using GM-CSF, IL-4, and monocyte-conditioned medium. DC exhibited the typical morphology with thin cytoplasmatic processes and expressed high levels of MHC class II, CD86, and CD83 typical for mature DC. After sorting with the monoclonal antibody CD83, a cell population of more than 95% CD83 positive cells was obtained. The presence of the Ph translocation was analyzed in these cells, in PBMC, lymphoblastoid cell lines (LCL), and in phytohemagglutinin (PHA)-induced T blasts from the same patients by fluorescence in situ hybridization (FISH). In contrast to all other cells analyzed, the vast majority of DC (95.9 +/- 0.7%) displayed the Ph translocation, irrespective of disease stage or therapy. PBMC were predominantly positive for the Ph chromosome (67.6 +/- 7.3%), whereas only 11.4 +/- 1% of the B cells and 4.4 +/- 1.1% of the PHA blasts carried the Ph translocation. Using such leukemic DC as antigen-presenting cells, a primary CML-directed cytotoxic immune response in vitro was obtained, as shown by the specific recognition of Ph chromosome positive cells. We conclude that DC can be generated from blood progenitors of CML patients in vitro and exhibit, to a large extent, the Ph translocation. Such DC, which in a preliminary experiment have been able to induce a primary CML-directed cytotoxic immune response in vitro, might be ideal candidates for adoptive immunotherapy either by direct transfer of DC for in vivo generation of a T-cell response or by in vitro generation of CML-specific cytotoxic autologous or HLA-matched normal T-cell clones for use in vivo.

Generation of monocyte-derived dendritic cells from precursors in rhesus macaque blood

While the dendritic cells (DCs) of mouse and man have been extensively studied, until recently those of non-human primates remained poorly characterized. We present a method for generating large numbers of DCs from precursors in rhesus macaque blood, based on techniques developed for human blood. For 7 days, a T cell-depleted population of mononuclear cells was cultured in 1% human plasma with GM-CSF and IL-4, both to initiate DC differentiation and to inhibit macrophage development. On day 7, 50% of the culture medium was replaced with a monocyte-conditioned medium (MCM), which is required for the final maturation of the DCs into potent stimulators of the allogeneic MLR. Between 0.5 and 1.0×10 6 DCs can be generated from 20 ml of rhesus macaque blood. We compared these cytokine-generated DCs to the adherent macrophages present in the same cultures. Cytokine-generated DCs were considerably more potent at stimulating allogeneic T cells than adherent macrophages. Furthermore, the DCs had a distinct morphology and phenotype, with long processes, high levels of p55, and a characteristic perinuclear collection of intracellular CD68. In contrast, adherent macrophages expressed very low levels of p55, and high diffuse levels of CD68. Macaque DCs generated by this method may be useful in vaccine development, and for studies of SIV pathogenesis.

The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand.

A subset of CD4+CD11c-CD3- blood cells was recently shown to develop into dendritic cells when cultured with monocyte conditioned medium. Here, we demonstrate that CD4+ CD11c-CD3- cells, isolated from tonsils, correspond to the so-called plasmacytoid T cells, an obscure cell type that has long been observed by pathologists within secondary lymphoid tissues. They express CD45RA, but not markers specific for known lymphoid- or myeloid-derived cell types. They undergo rapid apoptosis in culture, unless rescued by IL-3. Further addition of CD40-ligand results in their differentiation into dendritic cells that express low levels of myeloid antigens CD13 and CD33.

Production of inflammatory markers by HepG 2 cells stimulated with monocyte conditioned media: the effects of corticosteroid and other immunosuppressants

Both C-reactive protein (CRP) and serum amyloid A protein (SAA) are determined as an indicator of inflammation and tissue damage. We found that CRP decreased extremely after administration of corticosteroid but SAA did not. However, the mechanism of the CRP decrease by corticosteroid therapy is unclear. In this study we have examined the effects of some immunosuppressive drugs and cytokines on the production of CRP and SAA by human hepatoma cells (HepG 2). A corticosteroid prednisolone did not enhance the production of CRP by HepG 2 cells but enhanced that of SAA, which indicate that prednisolone had no direct effect on the CRP production. Some immunosuppressants other than corticosteroids suppressed the SAA production but had no effect on the CRP production. IL-1 beta induced both CRP and SAA production but only in the co-presence of IL-6. A cytokine IL-6 induced the CRP production in the presence of IL-1 beta, but did not affect the constitutive production of SAA. Then we have examined the cytokine production by monocytes stimulated by lipopolysaccharide. Prednisolone inhibited the production of IL-1 alpha, IL-1 beta, IL-6 and TNF alpha.

Monocyte-Induced Downregulation of Nitric Oxide Synthase in Cultured Aortic Endothelial Cells

Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell-induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented by NG-nitro-L-arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200 x 10(3) per well)-dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin-and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1 beta neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenous L-arginine failed to improve NO release, suggesting that oxidant stress-induced inactivation of NO or limited substrate availability were not primarily responsible for the inhibiting effects of monocytes. Western blot analysis revealed reduced quantities of ecNOS in monocyte/endothelium cocultures, as well as in HAECs treated with monocyte-conditioned medium or TNF-alpha. Thus, adhesion of monocytes to endothelial cells and monocyte-derived secretory products downregulate steady state levels of ecNOS, an event associated with attenuated release of biologically active NO. This mechanism may potentially contribute to diminished endothelium-dependent and NO-mediated vasodilation in early atherosclerosis.

Generation of mature dendritic cells from human blood An improved method with special regard to clinical applicability

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34 + precursors and GM-CSF plus TNF-α. The other method makes use of the more abundant CD34 − precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day ‘maturation culture’ to the initial 6–7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes (‘veils’), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34 + progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig, cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8–3.3 × 10 6 mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83 + cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.

IL-10 production is enhanced in human T cells by IL-12 and IL-6 and in monocytes by tumor necrosis factor-alpha.

IL-10, an immunoregulatory cytokine produced by T cells and monocytes, inhibits the expression of inflammatory and hemopoietic cytokines as well as its own expression. To evaluate the regulation of IL-10 production by T cells and monocytes, we measured IL-10 levels by ELISA in supernatants of PHA-stimulated PBMC following depletion of either T cells or monocytes. IL-10 production was significantly down-regulated in both T cell- and monocyte-depleted PBMC compared with undepleted PBMC, and IL-10 production could be restored by the addition of monocyte-conditioned medium (supernatant of PHA-stimulated, T cell-depleted PBMC), suggesting that IL-10 production by T cells is regulated by a monokine(s) produced by activated monocytes. To further clarify the monokine(s) responsible for IL-10 induction, we stimulated monocyte-depleted PBMC, purified CD4+, and CD8+ T cells with PHA and measured IL-10 production by ELISA and semiquantitative reverse transcriptase-PCR following monokine(s) addition. Addition of IL-6 and IL-12 enhanced IL-10 production in monocyte-depleted PBMC in a dose-dependent and additive manner. Furthermore, anti-IL-6 and anti-IL-12 Abs neutralized the IL-10-inductive effect of monocyte-conditioned medium. Similarly, IL-12 and IL-6 induced IL-10 production by purified CD4+ and CD8+ T cells. With respect to regulation of IL-10 produced by monocytes, TNF-alpha was found to induce IL-10 production by resting as well as by LPS-stimulated purified monocytes/macrophages. Taken together, these findings suggest that IL-10 production by human T cells and monocytes is differentially regulated. IL-12 and/or IL-6 can induce the expression of IL-10 by PHA-stimulated T cells, whereas TNF-alpha induces IL-10 production by monocytes. Since IL-10 inhibits the production of IL-6, IL-12, and TNF-alpha, these results may indicate a potential mechanism of negative feedback regulation of the immune response.

Quantitative Effects of Peripheral Monocytes and Nerve Growth Factor on CNS Neural Morphometric Outgrowth Parametersin Vitro

Wound healing of the central nervous system (CNS) is a complex process involving interactions between cells from both the vascular and the neural environments, extracellular matrix proteins, and a cocktail of agonistic and antagonistic bioactive molecules. Vascular cells, particularly peripheral monocytes and macrophages, are believed to play an important role in organizing and mediating CNS tissue reactions subsequent to penetrating injuries that compromise the blood–brain barrier. Although many investigators have studied the effect of macrophages and microglia (resident brain macrophages) on neural outgrowth, little is known regarding monocyte effects. We have combined tissue culture, video microscopy, and digital image processing and analysis to quantify morphometric parameters of neurons exposed to monocyte secretory productsin vitro.The experimental system developed is simple in design but provides a quantitative understanding of cellular function and molecular mechanisms and has the ability to both study processes of graded complexity and relate cellular function to overall systems behavior. We evaluate the efficacy of the experimental model developed by measuring morphometric parameters of human neural cells (hNT cell line) in the presence of nerve growth factor (NGF). Results suggest that monocyte-conditioned media (MCM) increases neuron outgrowth parameters, such as neuritic output, mean arbor output, neurite branching, and effective cell diameter. Moreover, we show that the bioactive factor present in MCM is not IL-1 and the activity of the factor with respect to neural outgrowth is between that of 10 and 100 ng/ml NGF.

Effect of pentoxifylline in rat and swine models of hepatic fibrosis: role of fibroproliferation in its mechanism

Fibroproliferation was studied in two animal models of liver disease. Oral feeding of yellow phosphorus to pigs reproducibly results in fibrosis after 8 weeks of feeding, extensive fibrosis after 12 weeks and cirrhosis after 16 weeks of yellow phosphorus. Bile duct ligation was used to induce cirrhosis in the rat. Fibroproliferation was assessed as uptake of tritiated thymidine into fibroblasts which had been incubated with monocyte-conditioned medium obtained from monocytes of pigs treated with yellow phosphorus or bile duct-ligated rats and compared to the corresponding controls. Fibrosis was assessed by collagen content of liver sections obtained from the two animal models. The collagen content was determined by quantitation of Sirius red/Fast green-stained liver sections. In both animal models collagen content was significantly elevated at the conclusion of the treatment. Collagen content of liver sections of yellow phosphorus-treated animals were elevated (40 ± 2.7, n = 15) compared to mineral oil-treated controls (23 ± 1.2, n = 12) and collagen levels in the bile duct-ligated rat model liver sections were elevated (31.2 ± 1.6, n = 6) compared to sham-operated controls (21.6 ± 0.7, n = 6). The results of the fibroproliferation assay indicate that monocytes obtained from pigs treated with yellow phosphorus produce fibroproliferative factors during the development of fibrosis. This is in contrast to the bile duct-ligated rat model where no differences were observed in the production of fibroproliferative factors in the bile duct-ligated rats compared to sham operated controls suggesting that this may not be a key event in this model of fibrosis. Pentoxifylline treatment of the yellow phosphorus induced swine model of hepatic fibrosis has been associated with a marked improvement in fibrosis. In this study treatment of fibrotic pigs with pentoxifylline was associated with an improvement in liver function tests, a reduction of collagen content of liver sections, and reduction in fibroproliferation in pigs receiving yellow phosphorus treatment. Fibroproliferative factors were produced during the development of fibrosis in the swine model of fibrosis and their effect was blocked by pentoxifylline administered in vivo. This is in contrast to the bile duct-ligated rat model where pentoxifylline treatment was not associated with improvement in liver function tests or reduction of collagen content of liver sections and did not alter the fibroproliferative activity of monocyte-conditioned media. Taken together these results suggest that fibroproliferation and increased synthesis of collagen are key events in the yellow phosphorus-induced pig model of hepatic fibrosis and that the action of pentoxifylline in this animal model is likely to be related to its effects on fibroproliferation with a subsequent effect on collagen production. This is in contrast to the bile duct-ligated rat model where the results from this study suggest that fibroproliferation per se does not appear to be a key event and where pentoxifylline treatment does not alter fibroproliferative activity nor alter the course of fibrosis in this animal model.

Human vascular smooth muscle cell-monocyte interactions and metalloproteinase secretion in culture.

Degradation of the atherosclerotic plaque extracellular matrix could destabilize the lesion, rendering it more prone to rupture. Both macrophages and vascular smooth muscle cells (SMCs) are potential sources of matrix metalloproteinases (MMPs), secreted enzymes that can digest vascular matrix. We explored interactions between human vascular SMCs and human monocytes that result in the secretion of interstitial collagenase (MMP-1) and stromelysin (MMP-3). Monocytes alone or those treated with SMC-conditioned media did not secrete these metalloproteinases as detectable by Western blot analysis. SMCs increased secretion of both MMP-1 and MMP-3 greater than 20-fold when cocultured with monocytes or when treated with monocyte-conditioned media. Addition of macrophage colony stimulating factor (< or = 1000 U/mL) to cocultures of monocytes and SMCs did not affect metalloproteinase secretion. Recombinant interleukin (IL)-1 receptor antagonist inhibited MMP-1 and MMP-3 induction in SMC cultures treated with monocyte-conditioned media (94% and 96% reduction, respectively), while a neutralizing antibody to tumor necrosis factor-alpha had no significant effect on metalloproteinase secretion. In contrast to the induction by monocyte-conditioned media of MMP-1 and MMP-3 secretion by SMCs, monocyte-conditioned media did not increase secretion of 72-kD gelatinase (MMP-2). Thus, monocytes induce MMP-1 and MMP-3 secretion by vascular SMCs through an IL-1-dependent mechanism. This response of SMCs to a defined macrophage product may contribute to plaque destabilization by mononuclear phagocytes in the lesion.

Human Satellite Cell-Proliferation in Vitro Is Regulated by Autocrine Secretion of IL-6 Stimulated by a Soluble Factor(s) Released by Activated Monocytes

We previously showed that macrophages, besides their scavenger role, selectively induce rat myoblast proliferation in vitro by releasing soluble factors. In this paper we demonstrate a relationship between human-activated monocytes and increased human myoblast proliferation due to IL-6 autocrine secretion by satellite cells. Indeed in the supernatants of muscle cultures treated with activated monocyte-conditioned medium we show by means of an ELISA quantitation a higher autocrine secretion of IL-6 associated with increased myoblast proliferation. This suggests that a growth factor(s) secreted by activated monocytes stimulates IL-6 production by myoblasts and then regulates proliferation of satellite cells.