Additive interaction of gefitinib ('Iressa', ZD1839) and ionising radiation in human tumour cells in vitro.

Cultures of human carcinoma A-431, A-549 and HeLa cells were challenged with gamma-rays without or with concomitant exposure to gefitinib, a potent inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR). The outcome of treatment was determined from cell and colony count, cell cycle progression and DNA double-strand break formation and rejoining. Apoptosis was measured in parallel from hypodiploid DNA and using an annexin V assay. Gefitinib developed a cytostatic effect in all cell lines, with drug sensitivity correlating the level of EGFR expression. A weak cytotoxicity of gefitinib was observed in HeLa cells only, although the drug was unable to induce significant cell cycle redistribution in this cell line. In contrast, substantial G1 block and S-phase depletion was observed in A-431 and A-549 cells exposed to gefitinib. The drug brought about additive to subadditive interaction with radiation with regard to growth inhibition, clonogenic death and induction of apoptosis. Consistently, gefitinib did not hinder the rejoining of radiation-induced DNA double-strand breaks in any cell line. The results demonstrate that gefitinib may elicit cytotoxicity at high concentration, but does not act as a radiosensitiser in vitro in concomitant association with radiation.

Depletion of KIN17, a human DNA replication protein, increases the radiosensitivity of RKO cells.

The human KIN17 protein is a chromatin-associated protein involved in DNA replication. Certain tumor cell lines overproduce KIN17 protein. Among 16 cell lines, the highest KIN17 protein level was observed in H1299 non-small cell lung cancer cells, whereas the lowest was detected in MeWo melanoma cells. Cells displaying higher KIN17 protein levels exhibited elevated RPA70 protein contents. High KIN17 protein levels may be a consequence of the tumorigenic phenotype or a prerequisite for tumor progression. Twenty-four hours after exposure to ionizing radiation, after the completion of DNA repair, a co-induction of chromatin-bound KIN17 and RPA70 proteins was detected. Etoposide, an inhibitor of topoisomerase II generating double-strand breaks, triggered the concentration of KIN17 into punctuate intranuclear foci. KIN17 may be associated with unrepaired DNA sites. Flow cytometry analysis revealed that 48 h after transfection the uppermost KIN17-positive RKO cells shifted in the cell cycle toward higher DNA content, suggesting that KIN17 protein induced defects in chromatin conformation. Cells displaying reduced levels of KIN17 transcript exhibited a sixfold increased radiosensitivity at 2 Gy. The KIN17 protein may be a component of the DNA replication machinery that participates in the cellular response to unrepaired DSBs, and an impaired KIN17 pathway leads to an increased sensitivity to ionizing radiation.

Treatment of multiple sclerosis patients with interferon-beta primes monocyte-derived macrophages for apoptotic cell death.

Although interferon (IFN)-beta has shown a significant clinical benefit in multiple sclerosis (MS), its mechanism of action remains unclear. We found that IFN-beta treatment of patients with MS resulted in a significant increase in apoptotic cell death (measured by annexin V staining and nuclear fragmentation) of monocyte-derived macrophages, as compared with cells derived from patients before treatment. Stimulation of the cells with IFN-beta in vitro resulted in an even further increase of annexin V binding, as well as increased Fas (CD 95, APO-1) expression. However, no increased Fas expression, apoptotic monocytes, or monocytopenia were observed upon in vivo treatment. This indicates that IFN-beta does not deliver a death signal to monocytes but rather primes for subsequent macrophage apoptosis upon activation or differentiation.

Distinct experimental efficacy of anti-Fas/APO-1/CD95 receptor antibody in human tumors.

Ligation of the Fas receptor (FasR) is a key step in apoptosis induction. Using a series of human tumor cells (SNB19, SNB79, 143N2, and SHEP), we observed a distinct efficacy of human anti-FasR antibody with an apparent correlation with Fas cell surface antigen expression. In contrast, all cells studied expressed detectable FasR mRNA transcripts. For all anti-FasR antibody-sensitive tumor cells, we showed a similar efficacy of Mab according to dose fractionation and injection site. We showed that, when injected into nude mice bearing human osteosarcoma 143N2, neuroblastoma SHEP, prostatic cancer PAC120, and the two glioblastomas SNB19 and SNB79, anti-FasR Mab induces significant inhibition of the growth rate of 143N2, SHEP, and PAC120 tumors, but has no efficacy on SNB19 and SNB79 tumors, with a relationship between in vitro and in vivo sensitivity to anti-FasR antibody. Altogether, these results suggest the antitumor potential of anti-FasR antibody in human neoplasms.

Regulation of the cell cycle by p53 after DNA damage in an amphibian cell line.

In mammalian cells, the p53 protein is a key regulator of the cell cycle following DNA damage. In the present study, we investigated the function of p53 in the A6 amphibian cell line. Using various specific Xenopus p53 monoclonal antibodies, we showed that Xenopus p53 accumulates after DNA damage, including gamma and UV irradiation or treatment with adriamycin. Such accumulation is accompanied by an increase in the apparent molecular weight of the protein. This change was shown to be the result of a phosphorylation event that occurs after DNA damage. Accumulation of Xenopus p53 is parallel to a drastic change in the cell cycle distribution. Brief exposure to adriamycin or gamma irradiation induces reversible growth arrest, whereas long-term exposure to adriamycin leads to apoptosis. Taken together, these results indicate that p53 has a similar behaviour in frog cells and mammalian cells, and that it conserves two activities, cell cycle arrest and apoptosis.

Contribution of nitric oxide to the apoptotic process in human B cell chronic lymphocytic leukaemia.

B cell chronic lymphocytic leukaemia (B-CLL) is characterised by defective apoptosis that cannot be explained solely on the basis of the known chromosomal abnormalities. We and other have now reported that the leukemic cells spontaneously display the inducible isoform of nitric oxide synthase, iNOS. Inhibition of the iNOS pathway leads to increased apoptosis of the tumoral cells in vitro, indicating that the endogenous release of NO contributes to their resistance to the normal apoptotic process. The factors that induce the expression of iNOS in vivo in the leukemic cells are not yet identified. Yet, as interaction of B-CLL leukemic cells with bone marrow stromal cells promotes their survival, the involvement of adhesion molecules and integrins may be suspected. The engagement of CD23 stimulates iNOS activation in the tumoral cells, suggesting that in vivo interaction of CD23 with one of its recognised ligands may contribute to iNOS induction. A role for CD40-CD40 ligand interaction may also be hypothesised. The mechanisms involved in the anti-apoptotic role of NO are not fully understood, but may implicate the inhibition of caspase activity, hence the impairment of the Fas pathway. In addition, the mitochondrial membrane potential disruption appears to be a NO-sensitive step in the apoptosis cascade. The presence of a NOS displaying anti-apoptotic properties has now been recognised in different cell types, including various leukaemia. A better knowledge of the mechanisms governing the ultimate fate of NO, anti- versus pro-apoptotic would allow the development of new therapeutic approaches for the treatment of these diseases.

Exacerbating effect of vitamin E supplementation on DNA damage induced in cultured human normal fibroblasts by UVA radiation.

The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320-380 nm) (UVA). Cells were incubated in medium containing alpha-tocopherol, alpha-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2.-) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2.- reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.

Arrest of S-phase progression is impaired in Fanconi anemia cells.

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5',8-trimethylpsoralen (Me(3)Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me(3)Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis. Taken together, the above data suggest that, in response to damage induced by DNA cross-linking agents, the S-phase checkpoint is inefficient in FA cells. This would lead to accumulation of secondary lesions, such as single- and double-strand breaks and gaps. The prolonged time in G2 phase seen in FA cells therefore exists in order to allow the cells to remove lesions which accumulated during the preceding abnormal S phase.

Regulation of CD26/DPPIV gene expression by interferons and retinoic acid in tumor B cells.

Interferons (IFNs alpha, beta and gamma) and all trans retinoic acid (RA) have the ability to activate genes with GAS sites. We have found that the promoter of CD26/dipeptidylpeptidase IV (DPPIV) contains a consensus GAS site TTCnnnGAA located at bp-35 to -27, and computer analysis confirmed this sequence to be a putative Stat binding site. Consistent with this finding, we show that IFNs and RA rapidly enhanced CD26 gene and protein expression in chronic B lymphocytic leukemia (B-CLL) cells. Immunoblot analyses revealed that unstimulated B-CLL cells expressed detectable levels of serine/tyrosine-phosphorylated Stat1alpha, and RA and IFN-gamma treatment led to increased levels of tyrosine phosphorylation of Stat1alpha and its nuclear accumulation. As shown by electrophoretic mobility shift assay, RA and IFN-gamma increased the binding of a nuclear protein to the GAS-CD26 element. Shift-Western blotting identified Stat1alpha as the GAS-CD26 binding factor. Augmented levels of CD26 protein in malignant B cells cultured with IFNs or RA coincided with the enhancement of DPPIV activity. Taken together, our results are in favor of the IFN-/RA-mediated upregulation of CD26/DPPIV in B-CLL through the signaling pathway involving Stat1alpha and the GAS response element of CD26 promoter.

Loss of alpha5beta1-mediated adhesion of monocytic cells to fibronectin by interferons beta and gamma is associated with changes in actin and paxillin cytoskeleton.

INTRODUCTION: Modulation of the adhesive responses of monocytic cells may reflect their motility within the bone marrow and at sites of inflammation. Monocyte alpha5beta1 integrins mediate fibronectin-dependent adhesion. We previously showed that type II IFN-gamma reduces adhesiveness to fibronectin (Fn) whereas TGF-beta1 enhances cell attachment. Here, we investigate the role of type I IFNs (alpha, beta) on the adhesive capacity of monocytic cells. MATERIALS AND METHODS: The influence of IFNs on the human U937 cell line adhesion to fibronectin-coated surfaces was determined. The expression of integrins and cytoskeleton proteins was analyzed by FACS, Western blotting and/or fluorescence microscopy analyses. RESULTS: IFN-alpha did not affect cell adhesion to fibronectin. In contrast, IFN-beta, like IFN-gamma, abrogated U937 adhesion to fibronectin and antagonized TGF-beta1-mediated cell attachment to Fn. The impaired binding of IFN-beta- and IFN-gamma-treated cells to fibronectin was not due to reduced levels of alpha5beta1 integrins. IFN-beta and IFN-gamma re-organized filamentous actin, and such rearrangement differed from that observed in TGF-beta1-adhesive cells. U937 cells dominantly expressed 44 to 46 kDa paxillin forms and treatment with IFNs enhanced the number of 66 to 70 kDa forms of paxillin. CONCLUSION: Our data show that IFN-beta and IFN-gamma induced loss of monocytic adhesion to fibronectin associated with changes in actin and paxillin cytoskeleton, thereby pointing to a possible effect of these cytokines in monocyte trafficking.

Upregulation of CD38 gene expression in leukemic B cells by interferon types I and II.

The activation antigen CD38, which has NAD+ glycohydrolase activity in its extracellular domain, is expressed by a large variety of cell types. Few investigations into the regulation of CD38 expression by physiologic stimuli have been reported. As the CD38 promoter contains potential binding sites for interferon (IFN) regulatory factor-1 (IRF-1), we investigated the influence of IFN type I (alpha and beta) and type II (gamma) on CD38 gene expression of leukemic B cells. Using the IFN-responsive B cell line Eskol, we found by RT-PCR analysis a rapid time-dependent induction in CD38 mRNA (starting at 6 h) with each type of IFN. This induction was independent of protein synthesis, suggesting that CD38 gene activation does not require IRF-1 but is merely under direct transcriptional regulation by latent IFN-inducible factors. mRNA stimulation was followed within 24 h by induction of membrane CD38, which coincided with rises of CD38-specific ectoenzymatic activities, that is, NAD+ glycohydrolase, (A/G)DP-ribosyl cyclase, and cyclic ADP ribose hydrolase activities. IFN failed to induce or upregulate the other CD38-related ectoenzymes analyzed, that is, CD39, CD73, CD157, and PC-1. Similarly, treatment of leukemic cells of patients with B chronic lymphocytic leukemia (B-CLL) with IFN resulted in an increase in CD38 mRNA mirrored by plasma membrane upregulation of CD38 and NAD+ glycohydrolase activity. Further investigation in relation to CD38 gene activation and B-CLL behavior remains to be defined.

Mutant p53 proteins stimulate spontaneous and radiation-induced intrachromosomal homologous recombination independently of the alteration of the transactivation activity and of the G1 checkpoint.

We report here a systematic analysis of the effects of different p53 mutations on both spontaneous and radiation-stimulated homologous recombination in mouse L cells. In order to monitor different recombination pathways, we used both direct and inverted repeat recombination substrates. In each line bearing one of these substrates, we expressed p53 proteins mutated at positions: 175, 248 or 273. p53 mutations leading to an increased spontaneous recombination rate also stimulate radiation-induced recombination. The effect on recombination may be partially related to the conformation of the p53 protein. Moreover, p53 mutations act on recombination between direct repeats as well as between inverted repeats indicating that strand invasion mechanisms are stimulated. Although all of the p53 mutations affect the p53 transactivation activity measured on the WAF1 and MDM2 gene promoters, no correlation between the transactivation activity and the extent of homologous recombination can be drawn. Finally, some p53 mutations do not affect the G1 arrest after radiation but stimulate radiation-induced recombination. These results show that the role of p53 on transactivation and G1 cell cycle checkpoint is separable from its involvement in homologous recombination. A direct participation of p53 in the recombination mechanism itself is discussed.

Correlations between p53-protein accumulation, serum antibodies and gene mutation in colorectal cancer.

Only half of colorectal-cancer patients elicit serum antibodies in response to intratumoral p53-gene mutations. Our study was designed to compare cellular events (p53-protein accumulation and gene mutations) with the presence of circulating anti-p53 antibodies (p53-Ab). Thirty-five colorectal-cancer patients were studied for their intratumoral p53-protein accumulation and circulating p53-Ab. Tumour DNA was analyzed for genomic mutations in a sub-set of 28 patients. In all, 18 tumours (51.4%) were positive by immunohistochemistry, and 17 tumour extracts were shown to contain "mutant" conformation p53 protein, 16 of them being were concordant by both methods. Of the 28 tumours tested by DGGE, 16 contained alterations in p53 exons 5 to 8 (57.1%). Of 12 tumours without detectable mutations, 10 were "mutant"-conformation-negative by immunohistochemistry and ELISA. Paradiploid tumors presented more frequently wild-type p53 genes and were significantly less frequently immunohistochemistry- or p53-Ab-positive than polyploid tumors. Circulating p53-Ab were detected in the serum of 11 patients (31%). In 9/11 cases, a gene mutation was found in the corresponding tumour. Three of four mutations in exon 8 and 3/3 mutations in exons 5-6 were associated with p53-Ab, in contrast with only 3/9 mutations in exon 7. We found good agreement in the detection of p53-gene alterations by different methods. However, our data suggest that all gene mutations may not be equivalent in term of immunogenicity.

Constitutive expression of CD26/dipeptidylpeptidase IV on peripheral blood B lymphocytes of patients with B chronic lymphocytic leukaemia.

We have investigated the expression of the ectoenzyme dipeptidylpeptidase IV (DPP IV)/CD26 on lymphocytes obtained from patients with B chronic lymphocytic leukaemia (B-CLL) and compared it with healthy subjects. Using two-colour immunofluorescence analysis with CD26 and CD20 or CD23 monoclonal antibodies, CD26 was found undetectable on peripheral resting B-cells (CD20+ CD23-) from normal donors whereas it was expressed on B-cells activated in vitro with interleukin (IL)-4 and Staphylococcus aureus strain cowan I (CD20+ CD23+). The expression of CD26 on leukaemic B-cells (CD20+ CD23+) was clearly induced in 22 out of 25 patients examined. Consequently, induced levels of CD26 cell surface expression on either normal activated and malignant B-cells coincided with the enhancement of DPP IV activity detected on the surface of these cells. Reverse transcription polymerase chain reaction analyses showed that the transcript levels of the CD26 gene was higher in normal activated B-cells and B-CLL cells than in resting B-cells, suggesting that CD26 was expressed at the level of transcriptional activation. These observations provide evidence of the abnormal expression of DPPIV/CD26 in B-CLL which, therefore, may be considered as a novel marker for B-CLL. Further investigation in relation to CD26 expression and other B malignancies needs to be defined.

The pag gene product, a physiological inhibitor of c-abl tyrosine kinase, is overexpressed in cells entering S phase and by contact with agents inducing oxidative stress.

The human pag gene product is an inhibitor of the c-abl tyrosine kinase and belongs to a new family of proteins. We show here that higher levels of pag gene expression are observed following induction of proliferation and contact with compounds inducing oxidative stress such as diethyl maleate and sodium arsenate. A weaker overexpression is seen in a macrophage cell line using hydrogen peroxide or menadione as inducers. Pag gene expression increases in synchronized cells entering the S phase. This raises the possibility that elevated levels of pag counteract the cytostatic activity of abl. Treatment of growth arrested cells with diethyl maleate and sodium arsenate induces pag gene overexpression, independently of cell proliferation. Thus, enhanced pag gene expression occurs in two cellular events: proliferation and response to oxidative stress.

Delaying the onset of M phase in NIH 3T3 cells blocked in early S phase occurs via accumulating cyclin B1 and tyrosine-phosphorylated p34cdc2 in the nucleus.

An affinity-purified antibody (anti-Cdc2C) raised against the carboxy terminal sequence LDNQIKKM of p34cdc2 uncovered in NIH 3T3 cells a protein subpopulation, the location and the level of accumulation of which evolve during progression through the cell cycle: it first emerges inside the nucleus in late G1/early S phase and continues to build up principally in this location throughout S phase; a cytoplasmic expression then becomes apparent near the end of S phase, develops during G2 and sometimes prevails over the nuclear expression; it finally relocates to the nucleus in early prophase. We propose that a major part of this subpopulation would represent p34cdc2 molecules existing inside a complex with cyclin B1. NIH 3T3 cells arrested in early S phase with aphidicolin do not commit prematurely to mitosis which indicates that the regulatory pathway involved in preserving the temporal order of S and M phases is functioning in these conditions. Conjugated Western blot analysis and immunofluorescence microscopy showed that cyclin A, cyclin B1 and tyrosine-phosphorylated p34cdc2 continue to build up predominantly in the nucleus of the arrested cells. After release from the block, the cells rapidly reenter S and G2 phases and, concomitantly, cyclin B1 and tyrosine-phosphorylated p34cdc2 relocate to the cytoplasm before redistributing again in the nucleus in early prophase. These data would suggest that delaying the onset of M phase in NIH 3T3 cells in which the rate of DNA replication is reduced, is first ensured by a mechanism that prevents the cytoplasmic relocation of inactive p34cdc2/cyclin B1 complexes continually forming in the nucleus once the G1 period of mitotic cyclin instability is over.

Induction of macrophagic differentiation and cytokine secretion by IgG1 molecules in human normal monocytes and myelogenous leukemia cells.

A role for IgG molecules in the activation of human myelogenous leukemia cells was examined. When added to monoblastic (U937) leukemia cells, mouse (m)IgG1 produced a dose- and time-dependent inhibitory growth effect associated with the induction of morphological features characteristic of macrophage maturation, and enhanced surface expression of Mac-1/CD11b characteristic of monocyte development. A study of isotype dependency of mig indicated that the effect was specific for Ig molecules of the IgG1 and IgG2b subclasses, whereas IgG2a or IgM had no effect. In parallel to U937 cell maturation, a marked production of latent TGF-beta was observed in supernatants of leukemia cells cultured with mIgG1. Myeloblastic (HL-60) leukemia cell line similarly responded to mIgG1 or mIgG2b in induction of macrophage differentiation and in the absence of neutrophil differentiation. Human blood monocytes cultured in the presence of mIgG1, exhibited higher levels of IL-1 beta and IL-6 mRNAs associated with an increase in protein extracellular release, suggesting that the effect of mIgG1 on IL-1 beta and IL-6 production in human monocytes was mediated at both transcriptional and post-transcriptional levels. Monocyte activation by mIgG1 and mIgG2b was associated with increased cell surface expression of HLA-DR class II molecules. Human IgG1 (and to a lesser degree hIgG2), was also capable of inducing leukemia cell growth arrest and macrophage maturation whereas F(ab')2 fragments of mIgG1 were not as efficient as intact mIgG1 in blocking cell growth. Most importantly, mAbs reactive with Fc gamma RII (CD32-specific Abs 2E.1 and IV.3) blocked the effects of mIgG1 on leukemia cell proliferation. Taken together, these data indicate that binding of IgG1 molecules, possibly through Fc gamma RII, may generate an activation signal towards myelogenous leukemia cells and normal counterpart cells, ie monocytes, leading to induction of macrophage maturation and cytokine secretion.

Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein.

Homologous recombination plays an essential role in processes involved in genome stability/instability, such as molecular evolution, gene diversification, meiotic chromosome segregation, DNA repair and chromosomal rearrangements. p53 devoid cells exhibit predisposition to neoplasia, defects in G1 checkpoint and high genetic instability but a normal rate of point mutations. We investigated the effect of a p53 mutation, on spontaneous homologous recombination between intrachromosomal direct repeat sequences, in mouse L cells. In these cells, wild type for the p53 gene, we have overexpressed the mutant p53(175(Arg>His)) protein leading to a p53 mutant phenotype, as verified by the absence of a G1 arrest after gamma-irradiation. We show that the rate of spontaneous recombination is increased from five- to 20-fold in the mutant p53 lines. Moreover, this increase is observed in gene conversion as well as in deletion events. Our results provide new insights into the molecular mechanisms of genetic instability due to a defect of p53.

Synergistic effect of prolactin on IFN-gamma-mediated growth arrest in human monoblastic cells: correlation with the up-regulation of IFN-gamma receptor gene expression.

Interferon-gamma (IFN-gamma) stimulates the development of monocytic features in human myeloid precursors. Because transcriptional regulation of IFN-gamma and the pituitary hormone prolactin (PRL) has been described to involve common Jak-STAT pathways, we addressed here the question of whether PRL plays a role in monoblastic (U937) cell growth and macrophage maturation. In contrast to IFN-gamma, PRL did not affect U937 cell growth nor induction of differentiation as assessed by the unchanged cell surface expression of maturation markers CD11b and HLA-DR class II. However, PRL in synergy with IFN-gamma inhibited, in a time- and dose-dependence, proliferation of U937 cells without influencing their maturation induced by IFN-gamma. IFN-gamma and PRL both affected the expression of the IFN-gamma receptor (IFN-gamma R) gene by increasing IFN-gamma R mRNA levels. The rise in IFN-gamma R transcripts was accompanied by a low but significant release of IL-6 which has previously been shown to stabilize IFN-gamma R mRNA. Moreover, a transient increase in surface expression of IFN-gamma R was observed in U937 cells treated by IFN-gamma alone or in combination with PRL, whereas no apparent modulation of cell surface IFN-gamma R was observed in cells treated with PRL. Lastly, PRL did not induce transcriptional activation in IFN-gamma inducible IRF-1 and Fc gamma RI genes in U937 cells. Together, our data indicate that IL-6 secretion and increased expression of the IFN-gamma R gene correlate with U937 cell growth arrest induced by IFN-gamma and PRL, probably through a signaling mechanism which does not involve the Stat 1/IRF-1 pathway.

TGF-beta 1-stimulated adhesion of human mononuclear phagocytes to fibronectin and laminin is abolished by IFN-gamma: dependence on alpha 5 beta 1 and beta 2 integrins.

Monocyte migration within the extravascular space of inflamed tissues is controlled by adhesion molecules and inflammatory cytokines. In this study, we analyzed the capacity of TGF-beta 1 and IFN-gamma to regulate adhesion of human activated monocytes to fibronectin (FN) and to laminin (LM), two components of the extracellular matrix. When cultured in the absence of any of these two stimuli, human monocytes underwent "spontaneous activation" and adhered to both FN and LM. Adhesion to FN was inhibited in the presence of alpha 5 and beta 1 integrin blocking antibodies, whereas beta 2 blocking antibody blocked attachment to LM. Exogenous TGF-beta 1 increased the adhesive ability of monocytes to FN and to LM, respectively, linked to the increase of alpha 5 and beta 2 mRNA and protein synthesis levels. Moreover, an increase in alpha 5 expression at the monocyte cell surface was observed. In contrast, monocytes stimulated with exogenous IFN-gamma lost their capacity to bind to FN and this coincided with the down-regulation of surface alpha 5 expression which occurred at the posttranscriptional level of alpha 5 synthesis. Although IFN-gamma-treated monocytes also showed a decreased ability to adhere to LM, no alteration of beta 2 mRNA levels, beta 2 protein synthesis, and beta 2 cell surface expression was detectable, thus suggesting a modification of the functional state of surface beta 2 integrins. Furthermore, when stimulated with TGF-beta 1, IFN-gamma-pretreated monocytes reacquired the ability to bind to FN and LM. Conversely, IFN-gamma reduced adhesiveness to FN and LM of monocytes initially stimulated with TGF-beta 1. These in vitro adhesive-deadhesive responses of monocytes to TGF-beta 1 and IFN-gamma modulation may reflect mononuclear phagocyte motility within sites of inflammation.