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Autocrine Type I IFN and Contact with Endothelium Promote the Presentation of Influenza A Virus by Monocyte-Derived APC¹

Chunfeng Qu^*, Thomas M. Moran and Gwendalyn J. Randolph^2,*

^* Carl C. Icahn Institute for Gene Therapy and Molecular Medicine and Department of Microbiology, Mt. Sinai School of Medicine, New York, NY 10029

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purified monocytes infected with influenza A virus do not become mature dendritic cells (DCs) and they present viral peptides poorly to autologous memory T cells. In this study, we investigated whether influenza A-infected monocytes matured to DCs with a high capacity to stimulate T cells when they were infected with influenza A virus in a model tissue setting wherein they were cocultured with endothelium grown on a type I collagen matrix. Intercellular interactions with endothelium strongly promoted the Ag-presenting capacity of monocyte-derived cells infected with influenza A virus, and the heterologous coculture system also enhanced production of IFN- by monocytes in the absence of plasmacytoid cells. Production of IFN- in the presence of endothelium correlated with monocyte differentiation to mature DCs and their ability to stimulate proliferation and IFN- production by autologous T cells. Monocyte-derived cells that developed into migratory DCs promoted proliferation of influenza A virus-specific CD4⁺ and CD8⁺ cells, whereas those that developed into macrophages promoted proliferation of CD8⁺ T cells only. This onset of APC activity could be partially blocked with Ab to the IFN- receptor when monocytes were infected with UV-treated virus, but neutralizing this pathway was inconsequential when monocytes were infected with live virus. Thus, type I IFN and direct contact with endothelium promote development of influenza A virus-presenting activity in monocyte-derived cells in a setting in which this differentiation does not depend on plasmacytoid cells. However, when infected with live influenza virus, the role of type I IFN in mediating differentiation and Ag-presenting capacity is expendable, apparently due to other mechanisms of virus-mediated activation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The interplay between distinct cell types to form a cooperative functional unit is the definition of tissues and organ systems. In the immune system, interactions between APC and lymphocytes have been widely studied in vitro using purified cell preparations without introduction of a tissue context. Experimental animal models then permit translation of observations made in vitro to in vivo relevance. This sound approach is sometimes limited by variations among species, and interpretations in vivo can be rendered difficult by a high degree of complexity that does not allow one to easily reconstruct minimal functional units. Experimental approaches that recapitulate intercellular events that occur within simple model tissues serve as an important bridge between studies of single-cell types in isolation and in vivo models. An excellent example of the rewards of such systems is the discovery that Peyer’s patch lymphocytes promote differentiation of Caco-2 intestinal cells to M cells that gain the capacity to transport bacteria (1).

We have used a simple model tissue that focuses on how endothelial cells, an abundant cell type in all tissues but which are not regarded as immune cells per se, influence the differentiation of human monocytes. Our previous work (2), and that of others (3, 4), revealed that monocytes have the capacity to develop into migratory Ag-presenting dendritic cells (DCs)³ or macrophages during coculture with endothelium. Otherwise, most of what we know about how human monocytes differentiate and acquire Ag-presenting function for protection against microbial pathogens has been generated from studies in which homogeneous cultures of monocytes were pretreated with cytokine mixtures and then exposed to pathogens in vitro for analysis of Ag presentation to T cells. These studies have contributed greatly to the field of DC biology, but a number of questions remain about how pathogens and their vaccine formulations might be handled by APC within tissues. The use of exogenous cytokine treatments to first generate at least partly differentiated DCs (5, 6) generally bypasses the first likely physiologic step: the influence of the pathogen itself and its tissue context on the induction of the differentiation process.

Generation of effector T cells from naive and memory pools has been characterized using human pathogens like influenza A virus. Secretion of type I IFN (IFN- and IFN-), particularly in response to double-stranded viral RNA, is an expected outcome of viral infection, and type I IFN has a key role in innate and adaptive immunity to viruses (7). The influence of viral infection on DC development from precursors within a model tissue setting and an investigation of the relative role of type I IFN, however, has not been fully dissected. Although various immune cell types have markedly different capacities to secrete IFN-, by far the highest capacity to produce IFN- arises from the rare CD123⁺CD4⁺CD45RA⁺CD11c^- plasmacytoid cell (8). Thus, the cellular source of IFN- that might be required for DC development has been anticipated to come from this cell type. Type I IFN, in conjunction with GM-CSF, has been reported to rapidly generate DCs from monocytes (9), but whether this combination of cytokines participates in, is required for, or leads to optimal Ag presentation of viral Ags is not clear. A recent study observed that IFN- was necessary but not sufficient for differentiation of DCs from monocytes in the autoimmune disease systemic lupus erythematosus (SLE) (10), but it is not certain to what extent this differentiation response resembles pathways triggered by common viral infections. Whether viruses like influenza A can even drive differentiation particularly of freshly isolated monocytes to DCs has been uncertain, due to the lytic capacity of the virus in monocytes in vitro (11) and the apparently poor induction of Ag-presenting function after purified monocytes become infected (6, 12).

Since monocytes and macrophages are well known for strong degradative capacity that may limit retention of antigenic peptides for presentation, we wished to determine whether monocytes that interact with pathogens before developing into DCs could indeed process and present relevant pathogen-derived Ags effectively once differentiated. Using functional and phenotypic criteria, we therefore investigated the effect of influenza A virus infection on monocytes that were cocultured in a model tissue setting with endothelium. We measured induction of IFN- production by T cells stimulated by monocytes incubated under various conditions after infection with influenza A virus. Our results indicate that monocytes themselves produce sufficient levels of IFN- to promote their maturation, although type I IFN becomes an expendable factor in the differentiation process to DCs when monocytes are infected with live virus. The data also reveal that direct intercellular interaction between monocytes and endothelium plays a key role in the generation of optimal Ag presentation capacity following influenza A infection. Finally, we observe that live virus infection leads to increased Ag-presenting function compared with the use of partially inactivated UV-treated virus, but the relative augmentation observed with live virus is similar in monocyte-derived macrophages to that in the migratory monocyte-derived DC population. Whereas both DCs and macrophages that develop from monocytes in response to influenza A virus infection caused proliferation of CD8⁺ T cells and induced their capacity to produce IFN-, only monocytes that differentiated to migratory DCs provoked proliferation of CD4⁺ T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Influenza virus

Influenza virus A/PR/8/34 (PR8) was grown for 40 h in 9-day-fertilized eggs at 37°C. Allantoic fluid was harvested and stored at -80°C. Virus was titered using a Madin-Darby kidney cell infectivity assay, as reported elsewhere (13). Titered, active influenza A virus was diluted in PBS (1/4). For some experiments, the virus was partially inactivated using UV light at 209 mW/cm for 5–10 min or was fully inactivated by boiling at 100°C for 15 min (14).

PBMCs

Blood from adult healthy donors or umbilical cord blood was collected according to guidelines approved by the Internal Review Board of Mt. Sinai School of Medicine (New York, NY). PBMCs were isolated by density gradient centrifugation on Ficoll. Magnetic depletions of NK cells and T and B lymphocytes were conducted using anti-CD56, anti-CD3, and anti-CD19 mini-MACS (Miltenyi Biotec, Auburn, CA), respectively. Depletion of CD123⁺ cells was conducted by incubating PBMCs with anti-CD123 mAb (BD PharMingen, San Diego, CA), followed by incubation with FITC-conjugated anti-mouse IgG and removal by flow cytometric cell sorting (Mo-Flo; Cytomation, Fort Collins, CO).

Reverse transmigration assay

Reverse-transmigrated and subendothelial leukocytes were studied using previously described methods (2, 15). In brief, 8 vol of bovine type I collagen gels (Cohesion, Palo Alto, CA) were mixed with 1 volume of 10x medium 199 (M199) and 5 vol of 0.1 N NaOH, and 50 µl of the solution were dispensed into microtiter wells for polymerization. On the following day, second passage HUVECs were cultured on the polymerized collagen to confluence in M199 containing 20% heat-inactivated human serum (HIHS). HUVECs were then incubated for 2 days further before addition of PBMCs. In some experiments, HUVECs were seeded onto collagen gels that were polymerized in the presence of 0.0025% zymosan (ICN Pharmaceuticals, Costa Mesa, CA), a particulate preparation derived from yeast.

For virus infection, PBMCs were mixed at a concentration of 5 x 10⁷ cells/ml with UV light-inactivated influenza A virus (multiplicity of infection (MOI), 5), heat-inactivated virus (MOI, 5), or live virus (MOI, 0.5) and incubated at 37°C for 45 min. Free virus was then washed out in medium containing 5% HIHS. Then virally infected PBMCs diluted in M199 containing 5% HIHS were applied onto HUVEC cultures and incubated for 1.5 h. Next, cultures were washed thoroughly in M199 to remove nonmigrated cells from above the endothelium. Fresh culture medium containing 20% of HIHS was added. In some experiments, 1000 U of type I IFN (R&D Systems, Minneapolis, MN), neutralizing anti-GM-CSF receptor mAb (2 µg/ml; Maine Biotechnology, Portland, ME), or neutralizing anti-IFN- receptor (5 µg/ml; R&D Systems) were included in the medium.

Transwell cultures

CD3⁺CD19⁺CD56⁺-depleted PBMCs (mostly monocytes) were infected with UV-inactivated influenza A virus and cultured in the upper chamber of a Transwell with 0.4-µm pores (Costar, Cambridge, MA). Beneath the Transwell in the lower chamber, a confluent HUVEC monolayer was grown on collagen (see Fig. 6 diagram). In some experiments, a portion of the added monocytes were cocultured with endothelial/collagen gels in the lower chamber, while other monocytes were placed into and confined to the interior of the Transwell where they could not contact the HUVECs.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Direct interactions between monocyte and endothelium promote optimal maturation and T cell activation. A, Diagram of Transwell experiment. Monocytes (Mo) were cultured with endothelium/collagen constructs directly (a). Alternatively, some monocytes were prevented from interacting directly with endothelium by containment in a Transwell above the endothelium. In some samples, monocytes were added to the upper chamber of the Transwell (0.4-µm pore size) and in the lower chamber with the endothelium (b). After 2 days, monocyte-derived cells were recovered from either the upper Transwell (b or c) or the endothelial monolayer itself (a). Expression of HLA-DR, CCR7, and CD86 were analyzed independently (B). ELISPOT assays for detecting IFN--producing autologous T cells were also conducted (C).

CD3⁺CD19⁺CD56⁺-depleted PBMCs were cultured under nonadherent conditions in Teflon beakers for 2 days in M199 containing 20% HIHS. In some experiments, 50 ng/ml (500 U/ml) GM-CSF and 1000 U/ml type I IFN was included in the medium. In other experiments, conditioned medium from endothelial cultures coincubated with monocytes infected with live virus was collected after 48 h of culture, filtered, frozen, and then added to monocytes incubated in Teflon beakers.

Flow cytometry

Abs used for flow cytometric staining included mAbs to CD14, CD86, CD123, HLA-DR (all BD PharMingen), CD83 (Serotec, Oxford, U.K.), and control IgG2a mAb UPC10 (Sigma-Aldrich, St. Louis, MO). Cell surface staining with these Abs was detected with FITC-conjugated rabbit anti-mouse Ig (DAKO, Carpinteria, CA). Multicolor analysis was conducted using PE-conjugated HLA-DR (BD PharMingen) and biotin-conjugated anti-CD86 mAb was detected using streptavidin-conjugated allophycocyanin (Caltag Laboratories, Burlingame, CA). For intracellular type I IFN staining, PBMCs infected with influenza virus were cultured in suspension for 2 days. The cells in suspension were treated directly with GolgiStop (BD PharMingen) containing monensin (2 µl/ml) for 2 h. Cells were washed and stained for CD14, as described above. After washing, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD PharMingen) at 4°C for 20 min. Intracellular IFN- was detected with rabbit anti-human IFN- polyclonal Ab (R&D Systems). Normal rabbit IgG (Pierce, Rockford, IL) was used for control staining. After washing, the cells were labeled with FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). To detect the influenza A virus-specific nucleoprotein (NP) expression, transmigrated monocytes were retrieved 14 h after infection with influenza A virus. After cell surface staining for CD14 and permeabilization, intracellular influenza A virus was stained with FITC-labeled mouse anti-NP (US Biological, Swampscott, MA).

ELISPOT assay for IFN-

Nitrocellulose plates (96-well, MAIP S45; Millipore, Bedford, MA) were coated with 50 µl of anti-human IFN- mAb (clone 1-DIK; Mabtech, Nacka, Sweden) at 10 µg/ml in NaHCO₃ buffer (pH 9.6) overnight at 4°C. The wells were washed with RPMI 1640 and then blocked with RPMI 1640 containing 5% HIHS for 2 h at 37°C. Irradiated, reverse-transmigrated cells derived from HUVEC/collagen cultures containing or lacking zymosan in the collagen gel, or monocytes cultured in Teflon beaker, were used as stimulators. One to 2 x 10⁵ autologous CD3⁺ T cells, obtained from PBMCs depleted of CD14⁺, CD16⁺ and HLA-DR⁺ cells using magnetic beads (CD3⁺ T, >90%), cultured in RPMI 1640 containing 10% FCS for 2 days were used as responder cells. In cord blood cell experiments, naive T cells were positively selected again using CD45RA mini-MACS (Miltenyi Biotec). The ratio of stimulator cells to responder cells was 1:10. When adult human PBMCs were used, coincubation of these cells was conducted for 20 h to measure recall responses that previously developed from natural influenza A infections. When cord blood PBMCs were used, coincubation was conducted for 40 h to permit sufficient time for activation and differentiation of naive T cells. Then the plate was washed six times with PBS containing 0.05% Tween 20. One hundred microliters of biotin-conjugated anti-IFN- mAb (1 µg/ml; Mabtech) diluted in PBS/0.5% BSA was added, and the plate was incubated at room temperature for 2 h. After washing with PBS/Tween 20, a avidin-HRP complex (Vectastain Avidin-Biotin Complex reagent; Vector Laboratories, Burlingame, CA) was added and incubated at room temperature for 1 h. Then 3-amino-9-ethylcarbazole (Sigma-Aldrich) substrate was added to develop the spots. Spots were read using an ELISPOT Reader (Zeiss, Oberkochen, Germany). Only spots with a fuzzy border and red color were counted (16). In some experiments, spots were also evaluated by manual counting in a stereoscope. The two methods of evaluation resulted in similar scores.

T cell proliferation

Autologous T cells, obtained from PBMCs depleted of CD14⁺, CD16⁺, and CD45RA⁺ cells using magnetic beads (Miltenyi Biotec), and HLA-DR⁺ cells (Dynal Biotech, Great Neck, NY), were cultured in RPMI 1640 containing 10% of FCS for 2 days without adding any cytokines. Then the T cells were labeled with 10 µM CFSE at 37°C for 15 min in protein-free PBS. Influenza A virus-infected monocytes derived from reversed-transmigrated or subendothelial populations (endothelial cells were depleted) were cocultured with the CFSE-labeled T cells at a ratio of 1:5 for 5 days. Proliferated T cells were stained with mouse anti-CD8 or CD4 (BD PharMingen), biotin-labeled goat anti-mouse (Jackson ImmunoResearch Laboratories), followed by staining with streptavidin-conjugated APC (Caltag Laboratories). Proliferated cells were detected by CFSE dilution using FACS after gating out propidium iodide-positive cells (17).

ELISA

Human GM-CSF and human IFN- ELISA detection kits (R&D Systems) were used to quantify the cytokines secreted into cell supernatants.

RT-PCR

Cells were retrieved from collagen by using collagenase D digestion and whole RNA was extracted using TRIzol (Life Technologies, Grand Island, NY) according to the method recommended by the manufacturer. All samples were treated with RNase-free DNase (Life Technologies) for 1 h at 37°C. One microgram of total RNA was synthesized into cDNA using Omniscript reverse transcriptase (Qiagen, Valencia, CA) and oligo(dT) primer (Promega, Madison, WI). MxA gene expression was amplified using MxA forward 5'-CTGTGGCCATACTG CGAGGA-3', nt 7–26 and MxA-reverse 5'-ACTCCTGACAGTGCCTCCAA-3', nt 469–488 (480 bp) (18). Primers to detect the G3PDH gene product (402 bp) were used as an internal control.

Statistics

Statistical analysis was conducted using Student’s t test in Microsoft Excel (Redmond, WA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endogenous production of IFN- and GM-CSF after influenza A virus infection

Monocytes were infected with influenza A virus before their addition to endothelial collagen cultures. Infection of PBMCs with live influenza A virus PR/8 using a MOI of >1 caused high mortality (data not shown), as previously reported for monocytes (11). UV treatment of the virus eliminated the problem of high mortality. Moreover, a lower dose of live virus (0.5 MOI) also preserved the viability of monocytes and was therefore used in our experiments.

To assay whether and what conditions monocytes were productively infected with influenza A virus, we measured expression of influenza A virus-specific NP 14 h after infection of PBMCs. The cells were detected by FACS analysis after double staining for the monocyte marker CD14 and influenza A virus-specific NP (Fig. 1A). When the PBMCs were infected with live influenza A virus (MOI, 0.5), 22–28% of the monocytes expressed the influenza A virus-specific NP 14 h after virus infection. NP expression was undetectable when the monocytes were infected with UV-inactivated or heat-inactivated virus, indicating that these preparations were sufficiently inactivated to prevent viral replication.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Production of type I IFN and GM-CSF in cultures of influenza A-infected PBMCs and endothelium. A, Expression of influenza A virus-specific NP within transmigrated monocytes. PBMCs were infected with live (MOI, 0.5), UV-inactivated (MOI, 5), or heat-inactivated influenza A virus (MOI, 5) and applied onto HUVECs. Fourteen hours later, the transmigrated cells were retrieved and analyzed by FACS analysis after double staining for the monocyte marker CD14 and influenza A virus-specific NP. B, RT-PCR amplification of MxA mRNA. Amplified MxA PCR products (480 bp) were electrophoresed in 1.2% agarose gel and stained with ethidium bromide. Lane M, 100-bp ladder (Bio-Rad, Hercules, CA); lane 1, transmigrated monocytes infected with live influenza A virus; lane 2, transmigrated monocytes infected with UV-inactivated influenza A virus; lane 3, transmigrated monocytes infected with heat-inactivated influenza A virus; and lane 4, transmigrated monocyte without virus infection. C, PBMCs were infected with live, UV-inactivated, or heat-inactivated influenza A virus and cultured in suspension or added to HUVEC/collagen constructs to permit monocyte transmigration into the subendothelial matrix. IFN- was measured in the conditioned medium by ELISA after 48 h (representative of three experiments). D, Some PBMCs were depleted of CD123+ cells by flow cytometric cell sorting. These cells, and those not depleted, were infected with UV-inactivated influenza A and cultured in suspension or with endothelial cultures that contained (+Zym) or lacked (No Zym) zymosan particles in the subendothelial collagen matrix. GM-CSF and IFN- were measured in the supernatants by ELISA after 48 h (representative of three experiments). E, PBMCs infected with UV-inactivated influenza A virus were cultured for 2 days and then stained for CD14 and intracellular type I IFN. Two-color dot plots are also plotted as a histogram that shows staining with matched control Ab (C, dotted line) or type I IFN staining in the presence (+V, bold line) or absence (-V, thin line) of virus infection. Zym, zymosan; Sus, suspension; HI, heat inactivated.

The production of IFN- was first examined using virus-infected monocytes maintained in suspension or cultured with endothelium. As anticipated from MxA expression analysis, the brief UV treatment of the virus to render it replication-defective did not prevent accumulation of IFN- (Fig. 1C). Levels of IFN- were slightly increased after infection with live virus relative to the levels observed using UV-treated virus, but production of IFN- was essentially undetectable when heat-inactivated virus was used (Fig. 1C). GM-CSF was detectable only when virus-infected cells were cocultured with endothelium (Fig. 1D). Surprisingly, coincubation of influenza A virus-infected monocytes with endothelium cultured on collagen containing zymosan resulted in lower levels of GM-CSF than observed in cultures containing uninfected monocytes (Fig. 1D).

CD4⁺CD123⁺HLA-DR⁺CD45RA⁺ plasmacytoid cells produce very high levels of IFN- compared with other cells (8), but are very low in abundance relative to the number of monocytes. We investigated the extent to which the accumulation of IFN- in our cultures after influenza A virus infection could be explained by the presence of these cells. Depletion of CD123⁺ cells from PBMCs by flow cytometric cell sorting did not greatly affect the amount of IFN- secreted in influenza A virally infected PBMC/HUVEC cultures (Fig. 1D), in contrast to a significant drop in IFN- observed in PBMCs cultured in suspension in the absence of plasmacytoid cells (Fig. 1D). This result suggested that another cell type in the PBMC or endothelial population produced IFN- after influenza A virus infection.

Infection of endothelial cells only with influenza A, in the absence of monocytes, did not lead to measurable levels of IFN- in the supernatant. Since monocytes are the main cell type retained in endothelial cultures after a 1.5-h coincubation of HUVECs with PBMCs, they seemed a likely candidate for producing IFN-. Indeed, when PBMCs were cultured for 2 days after virus infection and transport of cellular secreted products was blocked with monensin, intracellular accumulation of IFN- was detectable in CD14⁺ cells, indicating that monocytes synthesized IFN- after influenza A virus infection (Fig. 1E). These data suggest that coculture of monocytes with endothelial/collagen constructs facilitates IFN- production from monocytes in the absence of plasmacytoid DCs after influenza A virus infection.

Maturation by monocyte-derived cells incubated with various forms of influenza A virus and cocultured with endothelium

CD56-depleted PBMCs were infected with UV-inactivated influenza A virus and applied onto endothelial cells grown on a type I collagen gel. In these cultures, monocytes that have the capacity to become mature DCs traverse the endothelium in the ablumenal-to-lumenal direction (reverse transmigration), mimicking some aspects of migration into lymphatic vessels. Monocyte-derived cells that remain in the subendothelial collagen are inherently less responsive to chemoattractants than the migratory DC fraction (C. Qu and G. J. Randolph, unpublished observation) and bear characteristics more similar to macrophages (2). The cell surface phenotype of both the reverse-transmigrated and subendothelial populations were analyzed by FACS. After virus infection, the number of reverse-transmigrated cells increased by 15 ± 2.3% (p 0.002) with a concomitant decrease in the number of subendothelial cells, as compared with the number of reverse-transmigrated cells recovered from unstimulated endothelial cultures receiving PBMCs that were not infected with influenza (data not shown). Viral infection prompted both reverse-transmigrated and subendothelial monocyte-derived cells to decrease expression of CD14 (Figs. 1E and 2A). Down-regulation of CD14 was independent of coculture with endothelium (Fig. 1E). Influenza A virus infection resulted in modestly increased expression of HLA-DR and more robust increases in CD86 (Fig. 2B). Reverse-transmigrated cells began to express CD83 (Fig. 2) and the chemokine receptor CCR7 (Fig. 2A). This maturation response was most evident when monocytes were infected with live virus, was also observed when monocytes were infected with UV-inactivated virus, but was not evident with heat-inactivated virus (Fig. 2B).

View larger version (50K): [in this window] [in a new window]   FIGURE 2. Maturation responses of monocyte-derived cells after infection with different forms of influenza A virus and coculture with endothelium. Monocytes infected with live virus, UV-inactivated, or heat-inactivated virus were applied onto confluent HUVECs and cultured for 2 days. Expression of costimulatory molecules and markers associated with mature DCs were analyzed in reverse-transmigrated (RT) and subendothelial (SE) monocyte-derived populations. A, Staining with isotype-matched control mAb is indicated by thin lines in the histograms; bold lines indicate staining for specific cell surface markers after influenza A virus infection; and shaded profiles represent levels of cells surface markers observed in the absence of influenza A virus infection. B, Shaded profiles indicate the level of cell surface markers observed in subendothelial cells and bold lines indicate the level in reverse-transmigrated cells.

Next, we addressed whether influenza A virus-infected monocytes could stimulate T cell activation. ELISPOT assays to detect T cells producing IFN- indicated that live influenza A virus more potently induced recall responses than did UV-inactivated virus (Fig. 3A). Compared with UV-inactivated virus, infection with live virus notably stimulated subendothelial monocyte-derived cells (mostly macrophages) to activate IFN- production in influenza A virus-specific T cells. Thus, after infection with live influenza A virus, reverse-transmigrated and subendothelial monocyte-derived cells stimulated adult peripheral blood T cells (recall response, Fig. 3A) with similar potency. In three experiments in which live and UV-inactivated virus was directly compared, ELISPOT responses measured after infection with UV-inactivated virus were 58 ± 6.4% lower than infection with live virus in subendothelial macrophages, but only 31.2 ± 7.2% lower in reverse-transmigrated cells (Fig. 3A). When the CD123⁺ plasmacytoid DC population was depleted from the PBMC fraction used to generate reversed-transmigrated cells, no difference in the induction of IFN--producing T cells was observed compared with nondepleted cells (Fig. 3B). When cord blood was used as a source of monocytes and CD45RA⁺-selected naive T cells, reverse-transmigrated cells were more potent at inducing IFN--producing T cells using either live or UV-inactivated virus (Fig. 3C), consistent with the superior role of DCs compared with macrophages in initiating primary immune responses. The magnitude of the CD45RA⁺ naive T cell response from cord blood was 10- to 20-fold lower than whole T cells from adult blood. This result is consistent with the likelihood that memory T cells are the majority of responders in unselected T cells population from adult blood.

View larger version (62K): [in this window] [in a new window]   FIGURE 3. Assessment of autologous T cell responses in coculture with influenza A-infected monocyte-derived cells. ELISPOT analyses to assess activation of IFN--producing T cells were conducted using adult human monocytes and autologous T cells to measure recall responses (A). B, In some experiments, ELISPOT assays were performed after CD123+ cell depletion from the PBMC fraction that gave rise to reverse-transmigrated (RT) APC. C, In other experiments, reverse-transmigrated cells were prepared from cord blood and then mixed with autologous CD45RA+ naive T cells to measure primary responses to influenza A virus. D, CFSE-labeled autologous CD45RA-depleted memory T cells were incubated with influenza A virus-infected or control reverse-transmigrated cells or subendothelial (SE) cells for 5 days. Proliferated T cells were then stained with CD4 and CD8 to track the phenotype of proliferated cells. Data are representative of three experiments.

Role of endogenous type I IFN in generating potent influenza A virus-presenting monocyte-derived cells

Because the factors that promote maturation of APC in this culture model are endogenous, we next addressed whether endogenous type I IFN was required for the differentiation observed. First, we used neutralizing anti-IFN- receptor Ab to block type I IFN signaling. The neutralizing Ab was added to monocytes before virus infection or before exogenous type I IFN was applied to monocyte/HUVEC cocultures. This Ab was kept in the cultures for the entire 2-day incubation with endothelium before collection of reverse-transmigrated and subendothelial monocyte-derived cells. Its effect on type I IFN signaling was monitored by analysis of MxA gene expression (Fig. 4A). This Ab blocked signals generated by exogenous type I IFN, indicating that it remained potently neutralizing for type I IFN through the assay period. Most of the signaling induced by UV-inactivated influenza A virus to generate accumulation of MxA mRNA was also blocked by the anti-IFN- receptor Ab. However, MxA mRNA accumulation did not decrease significantly when the neutralizing Ab was added to cultures containing monocytes infected with live influenza A virus (Fig. 4A). These data indicate that anti-IFN- receptor Ab fully neutralizes signaling to type I IFN, but that MxA gene can be activated by other mechanisms following infection with live influenza A virus.

View larger version (49K): [in this window] [in a new window]   FIGURE 4. Role of endogenous type I IFN in the differentiation of monocyte-derived cells to potent APC after infection with influenza A virus and coculture with endothelium. Anti-IFN- receptor-neutralizing Ab (5 µg/ml, A-IFN) or control Ab (goat IgG) was incubated with PBMCs before the cells were infected with live or UV-inactivated influenza A virus or before addition of exogenous type I IFN. The neutralizing Ab was also included in cultures of endothelium with monocytes for the entire 2 days of coculture, with replenishment of Ab daily. A, RT-PCR amplification of MxA mRNA accumulation measured after 24 h of monocyte/endothelial coculture in 20 or 25 PCR cycles. Lane M, 100-bp ladder (Invitrogen, San Diego, CA); lane 1, live influenza A virus infection; lane 2, live influenza A virus infection plus anti-IFN- receptor Ab; lane 3, UV-inactivated influenza A virus infection; lane 4, UV-inactivated influenza A virus infection plus anti-IFN- receptor Ab; lane 5, exogenous type I IFN (1000 U/ml); lane 6, exogenous type I IFN (1000 U/ml) plus anti-IFN- receptor Ab; and lane 7, no virus, no exogenous type I IFN was added. G3PDH mRNA amplification was used as internal control. Expression of HLA-DR and CD86 (B and C) and ELISPOT analysis of IFN--producing T cells (D) were assessed in reverse-transmigrated (RT) and subendothelial (SE) monocyte-derived cells that were infected with virus and treated with control Ab (bold line, +V), infected with virus and treated with anti-IFN- receptor Ab (thin line, V plus A-IFN), or not infected with virus (shaded, -V). Staining with isotype-matched control mAb is indicated by dotted line (Neg). The mean fluorescence intensity in B is also summarized in C (three experiments were averaged).

Role of endothelium in maturation of influenza-infected monocytes requires intercellular interactions

In general, monocyte differentiation responses after viral infection correlated with the production of IFN- (above data). However, the increases of costimulatory and MHC molecules and the stimulation of T cells to produce IFN- were less marked when influenza A virus-infected monocytes were cultured in standard tissue culture plates (Fig. 5A), in suspension (Fig. 5B), or with collagen gels lacking endothelium (data not shown). Because, under these conditions, the levels of IFN- were not greatly affected by coculture with endothelium (Fig. 1D), these data suggest that IFN- may promote maturation of monocytes to DCs, but that it is not likely sufficient, similar to findings from SLE patients (10). Moreover, these data suggest that coculture with endothelium greatly augments the differentiation and Ag-presenting capacity of influenza virus-infected monocytes.

View larger version (40K): [in this window] [in a new window]   FIGURE 5. Influence of coculture with endothelium on cell surface phenotype and T cell stimulatory capacity of monocyte (Mo)-derived cells infected with influenza A virus. A, Monocytes infected with UV-inactivated influenza A virus were cultured in standard tissue culture plates (TC) or cocultured with an endothelium/collagen matrix. The expression of HLA-DR and CD86 was analyzed after 2 days. Reverse-transmigrated (RT) cells were assayed separately from the subendothelial (SE) population. B and C, Conditioned medium (CM) was collected from endothelial cultures that were incubated with influenza A virus-infected monocytes and then this medium was added to monocytes (no virus infection) cultured on suspension culture plastic (shaded profiles) or with endothelial/collagen gels (bold lines). As control, monocytes were cultured in the same setting without addition of conditioned medium and were either infected by influenza A virus (+V) or left uninfected (-V). Two days later, expression of HLA-DR, CD86, and CD83 was quantified (B) or the cells were used as stimulators of autologous T cells to assess the number of IFN--producing colonies by ELISPOT (C). HI, Heat inactivated.

The possibility that other soluble cytokines besides GM-CSF accumulated in the endothelial cultures containing virally infected monocytes and accounted for the enhanced differentiation response seen in endothelial cultures was next tested. We pooled conditioned medium from endothelial cultures coincubated with live influenza A virus-infected monocytes and determined whether that medium could promote DC maturation (Fig. 5B) and T cell stimulation (Fig. 5C) as effectively as that observed when monocytes were allowed to interact directly with endothelium. This conditioned medium did not have the same capacity to enhance maturation as direct interactions of monocytes with endothelial cell cultures (Fig. 5, B and C). Thus, the factors that mediate the differentiation of monocytes infected with live virus and cocultured with endothelium appeared not to be strictly soluble. However, soluble factors that participate in maturation were present in the conditioned medium (such as IFN-), because the conditioned medium added to monocytes that were also allowed to interact with endothelium could replace the need for viral infection itself in mediating maturation (Fig. 5B).

Because some key soluble factors may be labile or be consumed before the 48-h point at which the conditioned medium was collected, we took additional approaches to examine whether the maturation-inducing effects of the endothelium were due to production of soluble factors (Fig. 6A diagram). We used Transwells with 0.4-µm pore size to separate influenza A virus-infected monocytes (in upper Transwell chamber) from endothelium grown on collagen (in lower Transwell chamber). In some wells, influenza A virus-infected monocytes were also added to the endothelial layer to determine whether soluble factors generated vis-á-vis interactions of monocytes with endothelium could promote full maturation of monocytes present in the upper chamber and not allowed to contact the endothelium (Fig. 6A). Because monocytes incubated in the Transwells could not be distinguished according to whether they would become reverse-transmigrated or subendothelial monocyte-derived cells, all monocytes incubated in the lower chamber were pooled and analyzed as a single population rather than separating into reverse-transmigrated or subendothelial fractions. After virus infection, expression of HLA-DR, CD86, and CCR7 (Fig. 6B) and T cell stimulation (Fig. 6C) was highest in monocyte-derived cells that were able to interact directly with endothelium. Some degree of enhanced expression of HLA-DR and CD86, as well as a slight increase in T cell activation, was observed in monocytes recovered from the upper Transwell when monocytes were also present in the lower well, compared with endothelium only in the lower well (Fig. 6, B and C). These data suggest that soluble factors generated from monocyte-endothelial interactions may act in trans to facilitate maturation of neighboring monocytes, but that maximal differentiation responses require direct interactions with the endothelium. Thus, direct interactions with endothelium and soluble factors that include, but may not be limited to, type I IFN apparently cooperate to promote APC activity in influenza A virus-infected monocyte-derived cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies concluded that monocytes do not present peptides from influenza A virus after infection or when exogenously loaded with influenza A virus-derived peptides (6, 12). The same studies also show that monocytes are driven to apoptosis by infection with influenza A (11) virus. The findings herein, however, suggest that, within tissues, monocytes may respond to infection by a viral pathogen by differentiating into competent APC. The difference between our findings and the previous work is the influence of the environment and the dose of the virus. Although apoptosis of monocytes occurs in response to infection with live influenza, this response appears to be limited to incubations of monocytes with virus at relatively high MOI. Using a lower titer of virus, monocytes do not apoptose and are therefore eligible to develop into potent APC. Moreover, whereas the differentiation of monocytes to effective influenza A virus-presenting cells fails when monocytes are cultured alone, their coincubation with endothelium in a three-dimensional tissue-like setting optimizes induction of the Ag-presenting capacity.

A model for APC activation within tissues

DCs continuously migrate from tissues into afferent lymph, but not all of these trafficking cells possess properties of fully activated, or mature, APC. Rather, many possess markers associated with immature DCs (19), and such cells have been reported to contain phagocytic inclusions that likely arise from apoptotic cells with tissues (20). This evidence suggests that DCs are involved in an ongoing sampling of tissues. A popular and intriguing model that may account for how DCs regulate the induction and character of immune responses suggests that the recognition of pathogens (21, 22), as well as other types of danger (22), promotes the activation of DCs, leading to the onset of a phenotype in which T cells become engaged for onset of potent effector function.

Although the models discussed by Medzhitov and Janeway (21) and Matzinger (22) are often interpreted to mean that DCs act as the sensors of pathogenic danger themselves (5, 23), this need not be the case. Other cells within tissues (22), after sensing danger themselves, may in turn promote maturation of DCs via intercellular interactions, such as with endothelium or secretion of signaling molecules. Moreover, some cells that are not strictly committed to the DC lineage, such as monocytes, may have the capacity to develop into Ag-presenting DCs after receipt of appropriate signals from their environment.

We have previously conducted studies that illustrate the potential of monocytes to become migratory DCs when cocultured with endothelium. Those monocytes with the capacity to become DCs migrate across cultured endothelium in the basal-to-apical direction (reverse transmigration), a process that bears some resemblance to migration via lymphatic vessels and may also mimic the reverse-transmigration DCs across vascular endothelium observed in vivo (2, 24, 25, 26). In this model, reverse transmigration is not dependent upon maturation, since in the absence of inflammatory or pathogenic stimulus, the migratory cells do not display features of mature DCs. In agreement with in vivo analysis of migratory DCs (19), the present studies on influenza A virus infection and previous work using LPS and zymosan (yeast particles) (2) suggest that it is the character, or maturation status, of the migratory monocyte-derived cells, not their migration per se, that is altered by pathogens. Thus, this culture model appears useful for dissecting how pathogens regulate the activation of DCs and their precursors within a tissue setting. Indeed, very little is known about how human DCs are induced to mature from precursors like monocytes within tissues, and this lack of knowledge poses a significant barrier to the design of practical and effective vaccines. The advantage of this model over that of inducing monocyte differentiation to DCs using exogenous cytokines is that the current model makes no assumptions that cytokines, like GM-CSF and IL-4 or GM-CSF and IFN- (9), are already present in the environment during a period in which monocytes encounter a pathogen within a tissue. Rather, the model permits an analysis of endogenous mediators of differentiation.

Role and source of type I IFN in presentation of influenza A virus by monocyte-derived cells

One obvious question to address, therefore, was an analysis of the role of type I IFN in the ability of influenza A virus-infected monocytes to stimulate T cell effector function. Although the present data argue for an important role for type I IFN in the process of monocyte maturation to Ag-presenting DCs and macrophages after influenza A infection, we were particularly unable to block maturation of migratory DCs by neutralizing the interaction of type I IFN with the IFN- receptor after infection with live influenza A virus. It is unlikely that this observation arises from lack of sufficient neutralizing activity or availability of the anti-IFN- receptor Ab in the cultures, because subendothelial monocyte-derived macrophages in the same cultures remained sensitive to its effects and control experiments indicated that type I IFN response genes (MxA) were not induced in the presence of the Ab. Mechanisms that promote maturation of DCs in response to influenza A virus infection independent of type I IFN require additional pursuit. Apparently, after live influenza A virus infection, monocyte-derived DCs in this system can be matured by other mechanisms that are equally effective as that stimulated by type I IFN. Toll-like receptor 3 has been identified as the receptor for dsRNA (27), but it is not clear whether this receptor may participate in recognition of fully packaged virus or whether its engagement could generate IFN-independent maturation programs. Previous evidence suggests that dsRNA can induce MxA independently of type I IFN (28). However, the same work also described full blockade of MxA expression by PR/8 strain influenza A-infected DCs derived from GM-CSF and IL-4-treated monocytes in the presence of neutralizing anti-IFN Ab (28), in contrast to the finding in our model. Possibly, freshly isolated monocytes infected with PR/8 influenza A strain have more flexibility in response to the virus than that are present in partially differentiated DCs.

Circulating CD123⁺ plasmacytoid cells are known to produce the highest quantities of IFN- in response to viruses (8), but these cells were not essential for generating type I IFN in monocyte/endothelial cultures. That plasmacytoid cells would be greatly outnumbered by monocytes, which also produced IFN-, may account for the relative lack of importance that the plasmacytoid cells seemed to have in promoting DC development from monocytes in response to influenza A virus infection, even though on a per cell basis plasmacytoid cells produce vastly more IFN- than monocyte-derived cells (8).

A role for type I IFN in monocyte differentiation to DCs has previously been reported, albeit in different systems and contexts than studied herein. Serum from patients affected by the autoimmune disorder SLE augments the Ag-presenting capacity of monocytes, and the presence of IFN- in the serum is critical (10). In agreement with our findings, monocytes from SLE patients themselves could make IFN- in the absence of plasmacytoid cells. The same study indicates that IFN- was required but not sufficient for differentiation of monocytes into potent APC (10). However, these investigators suggested that the critical second factor(s) was soluble components in autologous human serum. Our system employs culture in human serum routinely, albeit not necessarily autologous, but nonetheless we found that interaction with endothelium was more effective at inducing differentiation of monocytes to DCs than any conditions that, in the absence of endothelium, contained human serum along with endogenous or exogenous type I IFN. It is not known whether SLE is caused by a viral infection (29) or specifically whether the monocyte-derived DCs from these patients are infected with virus or simply respond to IFN- produced by other cells.

Role of endothelium in mediating Ag-presenting capacity of monocyte-derived cells

Initially, we hypothesized that the role of the endothelium in promoting the acquisition of Ag-presenting capacity was to secrete GM-CSF, since this cytokine has been reported to collaborate with type I IFN (both cytokines added exogenously) to generate DCs from monocytes (9) with a similar kinetics as observed in the endothelial cultures. However, we observed that endogenous GM-CSF levels were relatively low after viral infection of monocytes. Stimulation of monocytes with yeast-derived zymosan present in the subendothelial collagen, in contrast, triggered very high levels of GM-CSF. Furthermore, attempts to neutralize GM-CSF activity had little effect on the increased expression of costimulatory molecules by monocyte-derived cells or upon their ability to induce IFN--secreting T cells. Finally, experiments in which monocytes and endothelial cells were cocultured, but prevented from intercellular contact, indicate that soluble factors alone, generated in the endothelial cultures, could not account completely for the role of the endothelium in promoting monocyte differentiation to optimal influenza A virus-presenting cells. Overall, the data suggest that direct interactions between monocytes and endothelial cells are critical. Future studies will be required to elucidate the nature of this requisite intercellular interaction.

Presentation of live vs UV-inactivated virus

Immunization of mice with live vs inactivated influenza A virus in mice highlights the superiority of live viral infections in leading to induction of Th₁-polarized effector activity (30). In the coculture system studied herein, live influenza A virus always led to an increased number of IFN--producing, influenza A virus-specific T cells as measured by ELISPOT. However, the difference between live and UV-inactivated influenza A virus in reverse-transmigrating cells containing mature DCs was less substantial than the differences observed between the two forms of the virus in activating the presentation capacity of subendothelial monocyte-derived cells that apparently develop into macrophages. Live, but not UV-inactivated influenza A virus led to presentation by subendothelial macrophages that was equivalent to that observed in migratory DCs with regard to the number of IFN--producing T cells clones induced. However, activation of CD4⁺ T cells was largely restricted to the reverse-transmigrating DC population. Thus, DCs may be particularly more efficient than macrophages at activating CD4⁺ T cells, which at least in their naive state appear to have a higher threshold for activation then CD8⁺ naive T cells (31, 32). Furthermore, subendothelial macrophages remained more dependent upon type I IFN for development of Ag-presenting capacity than did reverse transmigratory DCs. Whereas macrophages are not likely to participate in the induction of primary responses, in contrast to DCs (31, 33, 34, 35, 36, 37), they may have a key role in mediating recall responses of memory T cells within infected peripheral tissues (37). Such a biological response is reasonable in light of the trafficking patterns of memory T cells, whose effector functions may be induced within peripheral tissues when sources of specific Ag are present there (38). This possibility is consistent with recent findings that, in contrast to priming, T cell effector activity in experimental autoimmune encephalitis requires monocyte chemoattractant protein 1 (CCL2) via its consequent attraction of macrophages to the CNS (39).

In summary, the model system presented herein serves as a bridge between a fully reductive approach to investigating host-pathogen interactions using purified cell types and in vivo approaches that are too complex to completely dissect. This model particularly provides a framework for examining endogenous mediators that determine how monocyte-derived cells may respond to pathogens in a tissue setting. To compliment approaches that examine responses to direct infection with virus, an interesting further step in the model will be to examine endogenous mechanisms that may mediate and promote maturation of monocytes for cross-priming of virally infected cells residing in the collagenous matrix.

	Acknowledgments

 
We thank Andres Hidalgo (Department of Medicine, Mount Sinai School of Medicine, New York, NY) for collecting the cord blood cells and we are grateful to Dr. Andrea Mikulasova for critical reading of this manuscript and Dr. Reinhold Förster (Hannover, Germany) for providing mAb tohuman CCR7.

	Footnotes

 
¹ This work was supported in part by an Investigator Award from the Cancer Research Institute and National Institutes of Health Grant AI49653 (to G.J.R.).

² Address correspondence and reprint requests to Dr. Gwendalyn J. Randolph, Carl C. Icahn Institute for Gene Therapy, Mt. Sinai School of Medicine, 1425 Madison Avenue, Box 1496, New York, NY 10029. E-mail address: gwendalyn.randolph{at}mssm.edu

³ Abbreviations used in this paper: DC, dendritic cell; SLE, systemic lupus erythematosus; M199, Medium 199; HIHS, heat-inactivated human serum; MOI, multiplicity of infection; NP, nucleoprotein.

Received for publication July 1, 2002. Accepted for publication November 12, 2002.

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