HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carta, L. Articles by Varesio, L. Search for Related Content PubMed PubMed Citation Articles by Carta, L. Articles by Varesio, L.

Engineering of Macrophages to Produce IFN- in Response to Hypoxia¹

Luca Carta^*, Sandra Pastorino^2,*, Giovanni Melillo, Maria C. Bosco^*, Stefano Massazza^* and Luigi Varesio^*

^* Laboratory of Molecular Biology, G. Gaslini Institute, Genoa, Italy; and Developmental Therapeutics Program Tumor Hypoxia Laboratory, Science ApplicationsInternational Corporation, National Cancer Institute, Frederick, MD 21702

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activation of murine macrophages (M) requires the collaboration of signals derived from the immune system and the environment. In this study, we engineered a murine M cell line to become activated in response to an environmental signal, hypoxia, as the sole stimulus. Hypoxia is a condition of low oxygen tension, occurring in several pathological tissues, which acts in synergy with IFN- to induce full M activation. We transfected the ANA-1 murine M cell line with a construct containing the IFN- gene controlled by a synthetic promoter inducible by hypoxia (HRE3x-Tk), and we characterized the cellular and molecular biology of the engineered M under normoxia or hypoxia. Engineered M in normoxia expressed basal levels of IFN- mRNA and protein that were strongly augmented by shifting the cells to hypoxia. Furthermore, they responded to the synthesized IFN- with induction of IFN-responsive factor-1 and 2'-5'-oligoadenylate synthase expression. Under normoxic conditions, the engineered M had a significant constitutive level of Ia Ags and Fc receptors. Hypoxia induced further augmentation of Ia and Fc expression. Finally, hypoxia induced inducible NO synthase expression, and subsequent reoxygenation led to the production of NO. In conclusion, the engineered M, which produce IFN- in an inducible manner, express new biochemical and functional properties in response to low oxygen environment as the sole stimulus, thereby circumventing the need for costimulation by other immune system-derived signals.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The process of macrophage (M)³ activation requires the collaboration of several signals classically defined as "priming" and "triggering," the former derived from the immune system, the latter comprising bacterial products or environmental factors (1, 2, 3). We have demonstrated that hypoxia, defined as exposure of the cells to low oxygen tension, is a potent environmental factor that can induce, together with the M-activating cytokine IFN-, the expression of the inducible NO synthase gene (iNOS) in murine M (4). Transcriptional activation by the hypoxia is mediated, at least in part, by hypoxia-inducible factor 1 (HIF-1), which binds to the hypoxia-responsive element (HRE) present in the promoter of several genes (for review, see Refs. 5, 6, 7). HIF-1 is a heterodimer composed by two basic helix-loop-helix proteins, HIF-1 and HIF-1. These proteins belonging to the PAS family contain a 300-aa PAS domain (PER, ARNT, SIM), defined by their presence in the Drosophila PER and SIM proteins and in the mammalian ARNT and AHR proteins, the expression of which is regulated by hypoxia (8). The HIF-1 subunit is also known as the aryl hydrocarbon receptor nuclear translocator, a dimerization partner for the aryl hydrocarbon receptor constitutively expressed in unstimulated cells. HIF-1 is the hypoxia-responsive subunit of HIF-1, and its expression is tightly controlled by the O₂ concentration. The half-life of HIF-1 is <5 min under normoxia, and hypoxia induces accumulation of the protein. HIF-1 is a sequence-specific transcriptional activator that recognizes the HRE DNA binding site containing the core sequence 5'-RCGTG-3'. Several HIF-1-inducible genes have been identified, including erythropoietin, glycolytic enzymes, vascular endothelial growth factor, and inducible NO synthase, all of which contain the HRE in their promoter. The HRE can be activated by signals other than hypoxia (9), some of which are biologically relevant such as the metabolite of tryptophan picolinic acid (10, 11, 12), and others have pharmacological applications, such as desferioxamine (13). Hypoxia can occur at different degrees in the body, ranging from the relatively modest decrease in oxygen tension associated with high altitude to the anoxia in the ischemic tissue, the former sufficient to induce the synthesis of erythropoietin, the latter promoting apoptosis. Hypoxia is a significant pathophysiological component of many cardiovascular, hematological, and pulmonary disorders; wound healing and fibrosis; inflammatory conditions; and tumor formation, where it has been associated with resistance to radiotherapy, malignant progression, and metastasis formation (14, 15). Conceivably, the hypoxic environment will concur to modify the phenotype of M in those areas in which the cells are exposed to immune system-derived signals such as IFN- (5).

Activated M are endowed with effector and immunoregulatory functions that are important for the host defense against microbial infections and tumor growth (1, 3, 16). Attempts to exploit the functions of activated M for therapeutic applications involve either the i.v. administration of high doses of M activators to patients or the adoptive transfer of ex vivo activated monocytes (17, 18, 19, 20). However, the toxicity of IFN- or of other M activators (21) has discouraged the direct injection of these molecules into patients, whereas the adoptive immunotherapy approach using monocyte-derived M demonstrated that the treatment was well tolerated but that there was little evidence of therapeutic effect (17, 22). Part of the difficulties connected with manipulation of M activity is due to: 1) the need for multiple activating signals and particularly for the immune system-derived stimulation with potential negative side effects; and 2) the generalized response, not confined to the tissue of interest, elicited by injection of cytokines or of other M activators.

We are developing a model system with engineered M that may circumvent these problems. We report here on the engineering of murine M cell lines to express activation-associated properties in response to an environmental stimulus, hypoxia, as the sole signal, thereby circumventing the need for immune system-derived signals. Specifically, we have generated a murine M cell line to express an exogenous IFN- gene under hypoxic conditions. We describe the biological and molecular features of these cells and discuss their potential applications.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and culture conditions

The mouse M cell line ANA-1 was established by infecting fresh bone marrow-derived cells from C57BL/6 mice with the J2 recombinant retrovirus, carrying the v-raf/v-myc oncogenes, and it was shown to display the phenotypic and functional features and the morphology of well-differentiated M (23). ANA-1 M were cultured in DMEM (ICN Biomedicals, Aurora, OH), supplemented with 10% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Celbio, Milan, Italy). Cells were maintained at 37°C in a humidified incubator containing 20% O₂, 5% CO₂, and 75% N₂. For hypoxic conditions, cells were cultured in an atmosphere controlled-culture chamber (Bellco Glass, Vineland, NJ) containing a gas mixture composed of 94% N₂, 5% CO₂, and 1% O₂.

Reagents

Mouse rIFN- (sp. act. 10⁷ U/mg) was purchased from Life Technologies (Gaithersburg, MD). An ELISA kit for IFN- was purchased from Genzyme (Cambridge, MA). Desferrioxamine and ferrous sulfate (purity, 99%) were purchased from Sigma Chemical (St. Louis, MO). The content of endotoxin in all the reagents was below the detection limit of 6 pg/ml, as determined by testing the chromogenic Limulus amebocyte lysate (BioWhittaker, Walkersville, MD).

Plasmids

pHRE3xTk-IFN- is shown in Fig. 1 and was generated from the pBLCAT2 plasmid, containing the CAT reporter gene under the control of an HSV-tk promoter fragment spanning from -105 to +51. Three tandem copies of the double-strand oligonucleotide 5'-AGTGACTACGTGCTGCCTAGG-3' (iNOS-HRE) were subcloned into the HindIII/BamHI sites of pBLCAT2, 5' upstream to the HSV-tk promoter (11). The construct was sequenced using Sequenase version 2.0 (U.S. Biochemical, Cleveland, OH). The fragment encompassing the three tandem copies of iNOS-HRE, the HSV-tk promoter fragment spanning from -105 to +51, and the chloramphenicol acetyltransferase (CAT) gene was digested in HindIII/KpnI sites, klenowed in the HindIII site, and subsequently inserted in the NruI/KpnI sites of the pcDNA3 vector. The CAT-SV40 pA fragment was then deleted by digestion into XhoI/XbaI sites, and the IFN- cDNA, previously subcloned in XhoI/XbaI sites of pBluescript vector, was subsequently inserted into the same sites. The fragment encompassing the three copies of iNOS-HRE was excised from the pHRE3xTk-IFN- by digesting with NruI/BamHI restriction enzymes, thus obtaining the pTk-IFN- control plasmid. The constructs were sequenced using Sequenase version 2.0.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. pHRE3x-Tk-IFN- vector. HRE3x, three tandem copies of the HRE from the iNOS promoter; TK, HSV-Tk minimal promoter; IFN, murine IFN- full-length cDNA; SV40, internal SV40 promoter for transcription of the neomycin gene; SV40 pA, polyadenylation site from SV40.

The transfectant M cell lines HRE-IFN-M and Tk-Contr-M were generated by electroporating ANA-1 cells with the plasmids pHRE3x-Tk-IFN- and pTk-IFN-, respectively. The cells were washed and resuspended in complete medium at 25 x 10⁶ cells/ml. Plasmid DNA (10 µg) was added, and the cell suspension was incubated for 10 min on ice, electroporated at 240 mV/975 µF (Bio-Rad (Hercules, CA) Gen Pulser II), and incubated for an additional 10 min on ice. The cells were washed twice in complete medium and plated onto 100-mm plates (Costar, Cambridge, MA). After 48 h culture, stable transfectants were selected in medium containing G418 at 1 mg/ml (ICN Biomedicals).

Northern blot analysis

Total cellular RNA was purified with the Trizol Reagent (Life Technologies) according to the manufacturer’s instructions. A total of 20 µg RNA from each sample was size-electrophoresed under denaturing conditions on a 1.2% agarose gel containing 2.2 M formaldehyde, blotted onto a Nytran membrane (Schleicher & Schuell, Keene, NH), and cross-linked by UV irradiation. Filters were incubated overnight at 42°C in Hybrisol hybridization solution (Oncor, Gaithersburg, MD). The blots were then hybridized with the ³²P-labeled probes and autoradiographed as previously described (10). The cDNA probes, specific for murine IFN-, iNOS, 2'-5'-OASE, and M-inflammatory protein 1 (MIP1), generously provided by Dr. Antonio Sica (Istituto Mario Negri, Milan, Italy), were radiolabeled with [³²P]dCTP (Amersham, Arlington Heights, IL) by using a random priming kit (Life Technologies). Equal loading of RNA was assessed by blot hybridization with -actin probe, kindly provided by Prof. Cecilia Garrè (Istituto di Biologia e Genetica, Facoltà di Medicina e Chirurgia, Università di Genova, Genoa, Italy), and mRNA was quantified by densitometric analysis of the bands.

Western blot analysis

Cells were lysed at 1 x 10⁸ cells/ml in lysis buffer (10 mM phosphate, 1% Triton X-100, 0.5% SDS, 100 mM NaCl, 0.5% sodium deoxycholate, 0.5% NaN₃) containing 1 mM [4-(2-aminoethyl)benzenesulfonyl-fluoride hydrochloride], 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin. After 10 min incubation on ice, the lysates were spun at 8000 x g at 4°C for 10 min. The supernatants were stored at -70°C and tested for IRF-1 contents. The protein concentration was determined with the Bio-Rad protein assay kit (Bio-Rad). Total protein (100 µg) was subjected to 10% SDS-PAGE and electrophoresed under reducing conditions; Western blot analysis was performed as described previously (24). Briefly, proteins were transferred to Immobilon-P membrane (Millipore, Bedford, MA) by electroblotting at 300 mA for 2 h at 4°C. The membrane was blocked with 5% nonfat dry milk for 2 h at room temperature and subsequently incubated with anti-mouse IRF-1 mAb (Santa Cruz Biotechnology, Santa Cruz, CA) (2 µg/ml) for 1 h at room temperature with gentle shaking. The membrane was then washed and incubated with HRP-conjugated protein A (Pierce, Rockford, IL). IRF-1 protein expression was determined with an enhanced chemiluminescent assay kit (Pierce).

Immunofluorescence analysis

M were washed with PBS supplemented with 2% FBS and 0.05% NaN₃, hereafter referred to as immunofluorescence buffer, and preincubated for 20 min at 4°C with the anti-mouse CD16/CD32 rat mAb purchased from PharMingen (San Diego, CA) to block Fc III/II receptor-dependent nonspecific staining. Cells were then incubated for 30 min at 4°C with FITC-conjugated monoclonal anti-mouse Ia^b or isotype control mouse IgG2a Ab (PharMingen, Hamburg, Germany). For the analysis of the Fc III/II receptor expression, cells were incubated with FITC-conjugated goat anti-rat IgG (Sigma) after CD16/CD32 incubation. Surface staining was analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Detection of cytokine release

Cells were cultured onto 15-cm plates at 10⁶ cells/ml and incubated with the appropriate stimulus. After incubation, cell-free supernatants were harvested and assayed for IFN- protein using a specific ELISA from Genzyme following the manufacturer’s instructions.

Nitrite assay

The accumulation of NO₂^- was taken as a parameter for NO production by M cell lines (24). Briefly, cell-free supernatants were incubated with the Griess reagent (25) for 10 min at room temperature, and OD was measured in a Diamedix BP-96 microplate reader at 550 nm (Delta Biologicals, Rome, Italy). The concentration of NO₂^- was determined by the square linear regression analysis of a sodium nitrite standard that was measured in each experiment. The limit of sensitivity of the NO₂^- assay was 1.5 µM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Engineering M to produce IFN- in response to hypoxia

We have previously shown that the synthetic promoter containing three copies of the HRE element and the Tk minimal promoter confers hypoxia inducibility to a reporter gene transiently transfected into the M cell line ANA-1 (11). To generate a M cell line producing IFN- under hypoxic conditions, we constructed the pHRE3x-Tk-IFN- vector expressing the murine IFN- gene under the control of the HRE3x-Tk promoter and containing the neomycin resistance gene as a selectable marker for stable transfection in the ANA-1 M cell line (Fig. 1). The cells were transfected by electroporation and selected in medium containing 1 mg/ml neomycin. The neomycin-resistant M (HRE-IFN-M) were tested for IFN- production in normoxic or hypoxic conditions. We found that HRE-IFN-M produced low but detectable amounts of IFN- (28.2 pg/ml) after culture under hypoxic conditions for 24 h, whereas IFN- level was below the detection limit in cells cultured under normoxic conditions (data not shown). Control cultures, Tk-Contr-M transfected with the same vector without the HRE sequence (pTk-IFN-), or the parental ANA-1 M cell line were negative for IFN- production either constitutively or in response to hypoxia. These data demonstrated that the synthetic HRE3x-Tk promoter was inducible by hypoxia in stable transfectants and that it could drive the expression of IFN- in the ANA-1 M cell line.

The HRE-IFN-M was passaged in vitro for 1 month to assess the stability of the phenotype, and we observed that the production of IFN- in response to hypoxia decreased with time in culture, suggesting the takeover of IFN- nonproducing cells in the bulk population. To circumvent this problem, HRE-IFN-M were cloned by limiting dilution, and several clones were tested for IFN- production. Of 22 clones tested, 10 produced >50 pg/ml IFN- in response to hypoxia, and they were used for subsequent studies. Although multiple clones were used in different experiments, a similar pattern of results was observed in most of them. For clarity, we will describe the activity of one representative clone (M10), and we will mention only the different response that occurred with other clones.

To characterize the production of IFN-, M10 clone was incubated under normoxic or hypoxic conditions for 6 or 24 h, and the supernatants were tested for their IFN- contents. The results are shown in Fig. 2. We found low but significant levels of IFN- in M10 cultured under normoxic conditions. However, the hypoxic condition caused a 4- to 6-fold increase in the release of IFN- protein in the supernatant. A comparable degree of induction was consistently observed in more than four experiments performed.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Secretion of IFN- by M10. M10 cells were plated in 150-mm tissue culture dishes at a concentration of 1 x 106 cells/ml and cultured under normoxic (Medium) or hypoxic (Hypoxia) conditions for 6 or 24 h. Supernatants were harvested and assayed for IFN- content by ELISA. Results are the average of the results obtained in four independent experiments in which each sample was tested in triplicate.

View larger version (65K): [in this window] [in a new window]   FIGURE 3. Expression of IFN- mRNA by M10. M10 cells were plated in 150-mm tissue culture dishes at a concentration of 1 x 106 cells/ml and cultured under normoxic (Med) or hypoxic (Hy) conditions for 0, 6, or 24 h. Total RNA was extracted and probed with mouse full-length IFN- or mouse -actin cDNA. Data are from one representative experiment of >20 performed.

Response of M10 to hypoxia-inducible IFN-

M expression of class II histocompatibility Ags is a prerequisite for the Ag-presenting activity of the cells, and it is exquisitely sensitive to induction by levels of IFN-. Ia gene transcription is followed by increased surface expression of Ia Ags and Ag-presenting activity.

M10 cultures were incubated for 24 and 48 h under normoxic or hypoxic atmosphere and then tested for Ia Ag expression by FACS analysis. As shown in Fig. 4, ANA-1 cells are negative for Ia expression (Fig. 4, histogram A; mean fluorescence intensity, 0.5 x 10¹), confirming previous results (23). Furthermore, culture in hypoxia or normoxia did not induce Ia expression or modulate the levels of Ia induced by stimulation with IFN- (data not shown). In contrast, M10 cells expressed a basal level of Ia Ags (Fig. 4, histogram B; mean fluorescence intensity, 1 x 10²). Interestingly, hypoxia induced a significant increase in Ia expression on the surface of M10 cells that was evident after 24 h of culture (Fig. 4, histogram C; mean fluorescence intensity, 6 x 10³) and more pronounced after 48 h (Fig. 4, histogram D; mean fluorescence intensity, 9.5 x 10³). The basal level of Ia Ag expression by different clones of engineered cells was variable but always clearly distinct from the background and inducible by hypoxia.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. IAb surface expression by M10. Parental ANA-1 or M10 were plated in 100-mm tissue culture dishes at a concentration of 1 x 106 cells/ml and stained with anti-IAb mAb-FITC after culture under the following conditions: A, Untreated ANA-1 at time 0; B, untreated M10 at time 0; C, M10 cultured for 24 h in hypoxia; D, M10 cultured for 48 h in hypoxia. The log fluorescence intensity is plotted against the cell number. Data refer to a representative experiment.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. FcR expression by M10 macrophages. M10 were plated in 100-mm tissue culture dishes at a concentration of 1 x 106 cells/ml, cultured for 24 h in normoxia (histogram A) or hypoxia (histogram B), and incubated with rat anti-CD16/CD32 mAb, followed by PE-goat anti-rat Ig. Cell staining was measured by FACS analysis. The log fluorescence intensity is plotted against the cell number.

IFN- induces gene expression through activation of various transcription factors including IRF-1 (27) that activate transcription after interaction with IFN- responsive enhancer. To study whether hypoxia induced IRF-1 protein, we cultured M10 for 0, 6, and 24 h in normoxic or hypoxic conditions and then measured IRF-1 expression by Western blot analysis. M10 cultured under normoxic conditions expressed basal levels of IRF-1 that were increased 2- to 3-fold by stimulation with hypoxia for 6 h (Fig. 6A). Similar levels of IRF-1 were expressed at 24 h. These results demonstrated that the expression of IRF-1 paralleled the constitutive and hypoxia-inducible IFN- production in M10. In the same experiment, we tested the parental ANA-1 M that were stimulated for 6 or 24 h with medium or 100 U/ml exogenous IFN- as a positive control (Fig. 6B). ANA-1 M did not express detectable constitutive levels of IRF-1, but they responded to IFN- with IRF-1 induction that reached plateau levels in 6 h of stimulation.

View larger version (55K): [in this window] [in a new window]   FIGURE 6. Induction of IRF-1 protein. M10 or ANA-1 cells were plated in 150-mm tissue culture dishes at a concentration of 1 x 106 cells/ml. M10 were cultured under normoxic (Med) or hypoxic (Hy) conditions for 0, 6, or 24 h (Fig. 6A); ANA-1 were cultured in the presence of medium or 100 U/ml IFN- for 0, 6, or 24 h (Fig. 6B). The cells were tested for IRF-1 or -actin protein expression by Western blot. The results shown are from one representative experiment of three performed.

View larger version (69K): [in this window] [in a new window]   FIGURE 7. 2-5-OASE mRNA expression. A, Results of an experiment in which M10 cells were plated in 150-mm tissue culture dishes at a concentration of 1 x 106 cells/ml, cultured under normoxic (Med) or hypoxic (Hy) conditions for 0, 6, or 24 h, and tested for 2-5-OASE mRNA expression. RNA loading was checked by filter hybridization with mouse -actin probe. B, Results of a representative experiment in which ANA-1 cells were plated in 150-mm tissue culture dishes at a concentration of 1 x 106 cells/ml; cultured under normoxic (Med) or hypoxic (Hy) conditions for 0, 6, or 24 h in the presence or absence of IFN- (100 U/ml); and tested for 2-5-OASE mRNA expression. RNA loading was tested by evaluation of 28S and 18S rRNA, stained with ethidium bromide.

Functional response of M10 to hypoxia

We have previously reported that hypoxia is a costimulus with IFN- for iNOS mRNA expression in ANA-1 M and murine peritoneal M (11). Because hypoxia and IFN- exert synergistic effects, we hypothesized that hypoxia alone could be fully capable to drive iNOS mRNA expression in M10 cells by inducing IFN- and synergizing with it.

M10 clone and ANA-1 cells were cultured for 0, 6, and 24 h under hypoxic or normoxic conditions and tested for iNOS mRNA expression. Very low expression of iNOS mRNA was observed in M10 cells cultured in normoxia for 6 h and almost undetectable after 24 h culture (Fig. 8). In contrast, culture of M10 in hypoxic atmosphere determined up to an 8-fold increase at 6 h and a 10-fold increase at 24 h in iNOS mRNA. iNOS mRNA expression was not observed in parental ANA-1 M cultured in normoxic and hypoxic conditions at all the time points checked (data not shown).

View larger version (115K): [in this window] [in a new window]   FIGURE 8. Hypoxia induces iNOS mRNA expression in M10. M10 cells were plated in 150-mm tissue culture dishes at a concentration of 1 x 106 cells/ml and cultured under normoxic (Med) or hypoxic (Hy) conditions for 0, 6, or 24 h. Total RNA was extracted and probed with mouse iNOS cDNA or mouse -actin probe. Data are from one representative experiment of three performed.

View larger version (22K): [in this window] [in a new window]   FIGURE 9. NO production by M10. M10 cells were cultured for 2 days in normoxia (Normo), hypoxia (Hypo), or a combination of the two, as detailed. Cell-free culture supernatants were then harvested and tested for NO2- content. Data are from one representative experiment of three performed. Results are the average of the results obtained in four independent experiments in which each sample was tested in triplicate.

View larger version (74K): [in this window] [in a new window]   FIGURE 10. Expression of MIP1 under hypoxic conditions. ANA-1 and M10 (Mf10) cells were plated in 150-mm tissue culture dishes at a concentration of 1 x 106 cells/ml and cultured for 18 h under normoxic (Med) or hypoxic (Hy) conditions, in the presence or absence of IFN- (100 U/ml). Total RNA was extracted and probed with mouse MIP1, iNOS, or -actin cDNA. Data are from one representative experiment of three performed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Exposure of M10 cells to hypoxia caused a substantial increase in IFN- mRNA, readily followed by protein secretion and by triggering of IFN--dependent responses. The induction of IRF-1, a transcriptional activator involved in the regulation of both IFN- and IFN--inducible genes (27, 28), is consistent with the global response to IFN- induced by hypoxia.

Hypoxia augmented the Fc receptor within the first 24 h of treatment. The induction of class II Major histocompatibility Ags (Ia) in M10 cells followed a delayed kinetics leading to high levels of Ia expression at 48 h of exposure to hypoxia, similar to that observed in parental cells exposed to exogenous IFN-. Thus, the Ag-presenting functions and the Ab-dependent activities of M10, strictly controlled by the exogenous cytokines in conventional cells, were fully inducible by the sole environmental stimulation in M10 cells.

The murine M10 cell line ANA-1 expressed the dsRNA-dependent enzyme 2'-5'-OASE gene after stimulation with IFN-, as previously reported (29). 2-5-OASE is implicated in the biological response to IFN- (28), and the connection with the tumoricidal activity of M10 was suggested (30). M10 cells responded to hypoxia with augmentation of 2-5-OASE mRNA expression, indicating that the triggering of the dsRNA-dependent enzyme pathways occurs in these cells under hypoxic conditions. We noticed that hypoxia caused some decrease in the inducibility of 2-5-OASE mRNA by IFN- in the parental ANA-1 cell line. Our results indicated that the induction of IFN- by hypoxia followed by 2-5-OASE mRNA augmentation prevailed over the possible inhibitory effects of the stimulus.

M10 cells respond to hypoxia with accumulation of iNOS mRNA because IFN- acts in synergy with hypoxia in activating the murine iNOS promoter and in inducing transcription and translation of the gene (11). However, iNOS cannot metabolize arginine to citrulline under low oxygen tension, and NO is not produced under these conditions by normal M, parental M cell lines (11), or M10 cells. The production of NO by M10 can be reconstituted by shifting the cultures to normoxic conditions. Thus, the full effector responses, classically defined as NO-dependent activities, of M10 will be exerted in response to a shift in oxygen tension as seems to occur in the ischemia-reperfusion system. In the latter situation, NO-dependent tissue damage is evident during reperfusion but not during ischemia (4, 31).

The Tk minimal promoter is not inert but triggers a limited gene expression. In fact, M10 produce constitutively some IFN- mRNA and protein and respond to it, as shown by the significant levels of Ia, IRF-1, and 2-5-OASE expression under normoxic conditions. Hence, normoxic M10 differ from parental macrophages because they are not resting but they manifest a preactivation stage, also referred to as "primed," which renders them exquisitely susceptible to further activation by environmental stimulation (1). The lack of IFN- mRNA and protein and of IFN--inducible genes and functions in the parental ANA-1 cells argue against a potential role in this system of M-derived IFN- either constitutively or induced by hypoxia.

The biology of M in a hypoxic environment is currently being studied, and several genes are known to be induced by this stimulus (5). Although this study is focused on the M10, we measured the response of the parental ANA-1 cells stimulated with exogenous IFN- under hypoxic or normoxic conditions. We observed that among the responses tested, hypoxia alone did not induce gene expression, with the possible exception of MIP1, which was slightly but consistently augmented. Furthermore, hypoxia did not modulate gene induction by IFN-, with the possible exception of 2-5-OASE mRNA expression, which was slightly inhibited. Finally, we found that the constitutive expression of MIP1 by ANA-1 cells was decreased by IFN- under normoxic or hypoxic conditions, demonstrating also that one of the inhibitory activities of IFN- was insensitive to hypoxia. Taken together, these observations indicate that the majority of the M responses to IFN- tested thus far, whether positive or negative, are preserved in the hypoxic environment. These considerations are relevant to understand and appreciate the biological responses of the M10 macrophages that under hypoxic conditions will express a broad spectrum of IFN--inducible functions. Furthermore, the M10 cells did not produce MIP1 under hypoxic conditions because of the predominant inhibitory effects of IFN-. From a biological perspective, we can envision that M10, as opposed to normal M, in an hypoxic environment will produce a unique kind of inflammation characterized by fewer and/or different infiltrating leukocytes highly stimulated by IFN-. A more comprehensive analysis of the chemokine production is under way to provide a better definition of the M10 proinflammatory properties.

The choice of hypoxia as an inducer was stimulated by our long lasting interest in the response of M to low oxygen tension (4, 11, 12) and by the idea of creating a cellular M vector that could be of interest for gene therapy into pathological tissues. We have chosen to use the IFN- as a therapeutic gene, because it is a cytokine potentially active against tumors, as shown by various preclinical studies (19, 32) including transfer of IFN- gene into established brain tumors (33) and, at the same time, it is a macrophage activator. The major drawback for the clinical use of IFN- is its toxicity when given systemically (21). A M10 vector carrying an inducible IFN- gene could allow the delivery of the cytokine locally into the hypoxic tumor lesion and the achievement of therapeutic concentrations without major side effects because the secretion is restricted to the intercellular space. The secreted IFN- could exert antitumor effects by eliciting immunostimulatory activities of M10 themselves and/or of other infiltrating M, by inhibiting tumor proliferation, and by counteracting the proangiogenic activity (34) of hypoxia at the tumor site (14).

Other groups have suggested that macrophages can be used to deliver clinically useful genes to tumors. Griffiths et al. (35) used a hypoxia-regulated adenoviral vector to transduce human macrophages with either a reporter gene or a therapeutic gene encoding the human cytochrome P4502B6, which can activate a prodrug with antitumor activity. They demonstrated that infiltration of the transduced macrophages into tumor spheroids resulted in gene expression in response to hypoxia and tumor cell killing by activation of the prodrug. Their results suggest that these macrophage-based gene therapy systems can be applied to the human setting using primary cells and support the notion of the potential relevance of hypoxia-inducible cassettes for gene therapy. It will be interesting to compare the relative efficacy/toxicity of macrophages armed with prodrug-activating enzymes or cytokines for cancer gene therapy. Several shortcomings of the use of cellular, macrophage-based vectors for gene therapy can be overcome. The limited life span of the adenoviruses could be improved by using lentivirus- based vectors (36). Furthermore, murine M-like cells may be used for applications in humans, because xenogenic combinations for the treatment of glioblastoma have been already tested in human gene therapy protocols (37).

	Acknowledgments

 
We thank Chantal Dabizzi for secretarial assistance.

	Footnotes

 
¹ This work was supported by grants from the Italian Association for Cancer Research, "Progetto Finalizzato Biotecnologie," Centro Nazionale Ricerche, Telethon-Italy (A75), and the Italian Association Glycogenosis. S.P. is supported by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro.

² Address correspondence and reprint requests to Dr. Sandra Pastorino, Laboratory of Molecular Biology, G. Gaslini Institute, Largo G. Gaslini, 5, 16147 Genoa, Italy.

³ Abbreviations used in this paper: M, macrophages; Tk, thymidine kinase; HRE, hypoxia-responsive element; HIF, hypoxia-inducible factor; IRF, IFN-responsive factor; iNOS, inducible nitric oxide synthase; 2-5-OASE, 2'-5'-oligoadenylate synthase, MIP1, macrophage-inflammatory protein 1; CAT, chloramphenicol acetyltransferase.

Received for publication August 10, 2000. Accepted for publication February 20, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Varesio, L., I. Espinoza-Delgado, G. L. Gusella, G. Cox, G. Melillo, T. Musso, M. C. Bosco. 1995. Role of Cytokines in the Activation of Monocytes 55. Blackwell Scientific Publications, Cambridge, U.K.
2. Kopydlowski, K. M., C. A. Salkowski, M. J. Cody, N. Van Rooijen, J. Major, T. A. Hamilton, S. N. Vogel. 1999. Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. J. Immunol. 163:1537.[Abstract/Free Full Text]
3. Hamilton, T. A., Y. Ohmori, J. M. Tebo, R. Kishore. 1999. Regulation of macrophage gene expression by pro- and anti-inflammatory cytokines. Pathobiology 67:241.[Medline]
4. Melillo, G., S. Lynn, B. Taylor, A. Brooks, G. Cox, L. Varesio. 1996. Regulation of nitric oxide synthase expression in IFN-treated murine macrophages cultured under hypoxic conditions. J. Immunol. 157:2638.[Abstract]
5. Lewis, J. S., J. A. Lee, J. C. E. Underwood, A. L. Harris, C. E. Lewis. 1999. Macrophage responses to hypoxia: relevance to disease mechanisms. J. Leukocyte Biol. 66:889.[Abstract]
6. Semenza, G. L.. 2000. Hypoxia, clonal selection, and the role of HIF-1 in tumor progression. Crit. Rev. Biochem. Mol. Biol. 35:71.[Medline]
7. Ratcliffe, P. J., J. F. O’Rourke, P. H. Maxwell, C. W. Pugh. 1998. Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression. J. Exp. Biol. 201:1153.[Abstract]
8. Wang, G. L., B. H. Jiang, E. A. Rue, G. L. Semenza. 1995. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O₂ tension. Proc. Natl. Acad. Sci. USA 92:5510.[Abstract/Free Full Text]
9. Semenza, G. L.. 1998. Hypoxia-inducible factor 1 and the molecular physiology of oxygen homeostasis. J. Lab. Clin. Med. 131:207.[Medline]
10. Melillo, G., G. Cox, A. Biragyn, L. Sheffler, L. Varesio. 1994. Regulation of nitric oxide synthase mRNA expression by interferon and picolinic acid. J. Biol. Chem. 269:8128.[Abstract/Free Full Text]
11. Melillo, G., T. Musso, A. Sica, L. S. Taylor, G. W. Cox, L. Varesio. 1995. A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J. Exp. Med. 182:1683.[Abstract/Free Full Text]
12. Melillo, G., L. S. Taylor, A. Brooks, T. Musso, G. W. Cox, L. Varesio. 1997. Functional requirement of the hypoxia-responsive element in the activation of the inducible nitric oxide synthase promoter by the iron chelator desferrioxamine. J. Biol. Chem. 272:12236.[Abstract/Free Full Text]
13. Wang, G. L., G. L. Semenza. 1993. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 82:3610.[Abstract/Free Full Text]
14. Maxwell, P. H., G. U. Dachs, J. M. Gleadle, L. G. Nicholls, A. L. Harris, I. J. Stratford. 1997. Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc. Natl. Acad. Sci. USA 94:8104.[Abstract/Free Full Text]
15. Graeber, T. G., C. Osmanian, T. Jacks, D. E. Housman, C. J. Koch, S. W. Lowe, A. J. Giaccia. 1996. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors. Nature 379:88.[Medline]
16. Gendelman, H. E., P. S. Morahan. 1994. Macrophages in Viral Infections 157. IRL Press, Oxford.
17. Andreesen, R., B. Hennemann, W. Krause. 1998. Adoptive immunotherapy of cancer using monocyte-derived macrophages: rationale, current status, and perspective. J. Leukocyte Biol. 64:419.[Abstract]
18. Kaplan, J. M., Q. Yu, S. T. Piraino, S. E. Pennington, S. Shankara, L. A. Woodworth, B. L. Roberts. 1999. Induction of antitumor immunity with dendritic cells transduced with adenovirus vector-encoding endogenous tumor-associated antigens. J. Immunol. 163:699.[Abstract/Free Full Text]
19. Lei, H., D. W. Ju, Y. Yu, Q. Tao, G. Chen, S. Gu, H. Hamada, X. Cao. 2000. Induction of potent antitumor response by vaccination with tumor lysate-pulsed macrophages engineered to secrete macrophages colony-stimulating factor and interferon-. Gene Ther. 7:707.[Medline]
20. Nishihara, K., R. F. Barth, N. Wilkie, J. C. Lang, Y. Oda, H. Kikuchi, M. P. Everson, M. T. Lotze. 1995. Increased in vitro and in vivo tumoricidal activity of a macrophage cell line genetically engineered to express IFN, IL4, IL6, or TNF. Cancer Gene Ther. 2:113.[Medline]
21. Kurzrock, R., S. Rosenblum, A. Sherwin, A. Rios, M. Talpaz, J. R. Quesada, J. U. Gutterman. 1985. Pharmacokinetics, single-dose tolerance, and biological activity of recombinant -interferon in cancer patients. Cancer Res. 45:2866.[Abstract/Free Full Text]
22. Hennemann, B., A. Rehm, S. Kottke, N. Meidenbauer, R. Andreesen. 1997. Adoptive immunotherapy with tumor-cytotoxic macrophages: phase I trial to study the mobilization of blood monocytes with recombinant human granulocyte-macrophage colony-stimulating factor. J. Immunother. 20:365.
23. Blasi, E., D. Radzioch, S. K. Durum, L. Varesio. 1987. A murine macrophage cell line, immortalized by v-raf and v-myc oncogene exhibited normal macrophage differentiated functions. Eur. J. Immunol. 17:1491.[Medline]
24. Bosco, M. C., R. E. Curiel, A. H. Zea, M. G. Malabarba, J. R. Ortaldo, I. Espinoza-Delgado. 2000. IL-2 signaling in human monocytes involves the phosphorylation and activation of p59^hck1. J. Immunol. 164:4575.[Abstract/Free Full Text]
25. Cox, G. W., G. Melillo, U. Chattopadhyay, D. Mullet, R. H. Fertel, L. Varesio. 1992. TNF--dependent production of reactive nitrogen intermediates regulates IFN- plus IL-2 induced murine macrophage tumoricidal activity. J. Immunol. 149:3290.[Abstract]
26. Young, H. A., K. J. Hardy. 1995. Role of interferon- in immune cell regulation. J. Leukocyte Biol. 58:373.[Abstract]
27. Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, S. I. Koh, T. Kimura, S. J. Green, et al 1994. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 263:1612.[Abstract/Free Full Text]
28. Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67:227.[Medline]
29. Melillo, G., G. W. Cox, D. Radzioch, L. Varesio. 1993. Picolinic acid, a catabolite of L-tryptophan, is a co-stimulus for the induction of reactive nitrogen intermediate production in murine macrophages. J. Immunol. 150:4031.[Abstract]
30. Varesio, L., D. Radzioch, B. Bottazzi, G. L. Gusella. 1992. Ribosomal RNA metabolism in macrophages. Curr. Top. Microbiol. Immunol. 181:208.
31. Granger, D. N., R. J. Korthius. 1995. Physiology mechanisms of posthischemic tissue injury. Annu. Rev. Physiol. 53:311.
32. Forni, G., P. L. Lollini, P. Musiani, M. P. Colombo. 2000. Immunoprevention of cancer: is the time ripe?. Cancer Res. 60:2571.[Abstract/Free Full Text]
33. Fathallah-Shaykh, H. M., L.-J. Zhao, A. I. Kafrouni, G. M. Smith, J. Forman. 2000. Gene transfer of IFN- into established brain tumors represses growth by antiangiogenesis. J. Immunol. 164:217.[Abstract/Free Full Text]
34. Folkman, J.. 2000. Angiogenesis and angiogenesis inhibition: an overview. EXS 79:1.
35. Griffiths, L., K. Binley, S. Iqball, O. Kan, P. Maxwell, P. Ratcliffe, C. Lewis, A. Harris, S. Kingsman, S. Naylor. 2000. The macrophage-a novel system to deliver gene therapy to pathological hypoxia. Gene Ther. 7:255.[Medline]
36. An, D. S., R. P. Wersto, B. A. Agricola, M. E. Metzger, S. Lu, R. G. Amado, I. S. Chen, R. E. Donahue. 2000. Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34⁺ cells. J. Virol. 74:1286.[Abstract/Free Full Text]
37. Culver, K. W., Z. Ram, S. Wallbridge, H. Ishii, E. H. Oldfield, M. Blaese. 1992. In vivo gene transfer with retroviral vector producer cells for treatment of experimental brain tumors. Science 256:1550.[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Ricciardi, A. R. Elia, P. Cappello, M. Puppo, C. Vanni, P. Fardin, A. Eva, D. Munroe, X. Wu, M. Giovarelli, et al. Transcriptome of Hypoxic Immature Dendritic Cells: Modulation of Chemokine/Receptor Expression Mol. Cancer Res., February 1, 2008; 6(2): 175 - 185. [Abstract] [Full Text] [PDF]

				M. Puppo, M. C. Bosco, M. Federico, S. Pastorino, and L. Varesio Hypoxia inhibits Moloney murine leukemia virus expression in activated macrophages J. Leukoc. Biol., February 1, 2007; 81(2): 528 - 538. [Abstract] [Full Text] [PDF]

				M. C. Bosco, M. Puppo, C. Santangelo, L. Anfosso, U. Pfeffer, P. Fardin, F. Battaglia, and L. Varesio Hypoxia Modifies the Transcriptome of Primary Human Monocytes: Modulation of Novel Immune-Related Genes and Identification Of CC-Chemokine Ligand 20 as a New Hypoxia-Inducible Gene J. Immunol., August 1, 2006; 177(3): 1941 - 1955. [Abstract] [Full Text] [PDF]

				M. Puppo, S. Pastorino, G. Melillo, A. Pezzolo, L. Varesio, and M. C. Bosco Induction of Apoptosis by Flavopiridol in Human Neuroblastoma Cells Is Enhanced under Hypoxia and Associated With N-myc Proto-oncogene Down-Regulation Clin. Cancer Res., December 15, 2004; 10(24): 8704 - 8719. [Abstract] [Full Text] [PDF]

				M. C. Bosco, M. Puppo, S. Pastorino, Z. Mi, G. Melillo, S. Massazza, A. Rapisarda, and L. Varesio Hypoxia Selectively Inhibits Monocyte Chemoattractant Protein-1 Production by Macrophages J. Immunol., February 1, 2004; 172(3): 1681 - 1690. [Abstract] [Full Text] [PDF]

				B. Burke, A. Giannoudis, K. P. Corke, D. Gill, M. Wells, L. Ziegler-Heitbrock, and C. E. Lewis Hypoxia-Induced Gene Expression in Human Macrophages: Implications for Ischemic Tissues and Hypoxia-Regulated Gene Therapy Am. J. Pathol., October 1, 2003; 163(4): 1233 - 1243. [Abstract] [Full Text] [PDF]

B. Burke, S. Sumner, N. Maitland, and C. E. Lewis Macrophages in gene therapy: