EBM Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nimura, F.
Right arrow Articles by Tanaka, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nimura, F.
Right arrow Articles by Tanaka, Y.
Experimental Biology and Medicine 231:431-443 (2006)
© 2006 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Cross-Linking Cell Surface Chemokine Receptors Leads to Isolation, Activation, and Differentiation of Monocytes into Potent Dendritic Cells

Fumikazu Nimura*,{dagger}, Li Feng Zhang*, Kazu Okuma*, Reiko Tanaka*, Hajime Sunakawa{dagger}, Naoki Yamamoto{ddagger} and Yuetsu Tanaka*,1

* Department of Immunology and {dagger} Department of Oral and Maxillofacial Functional Rehabilitation, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan; and {ddagger} AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan

1 To whom requests for reprints should be addressed at Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Uehara 207, Nishihara-cho, Nakagami-gun, Okinawa 903-0215, Japan. E-mail: yuetsu{at}s4.dion.ne.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Monocytes express on the cell surface several kinds of chemokine receptors that facilitate chemotaxis followed by differentiation in target tissues. In the present study, we found that a large number of monocytes from peripheral blood mononuclear cells (PBMCs) tightly adhered to plastic cell culture plates precoated with a monoclonal antibody (mAb, clone T312) specific for human CCR5 but not an isotype control after overnight incubation. Soluble T312 did not induce such adhesion, indicating that cross-linking of CCR5 is required for the enhanced adhesion of monocytes. The adhesion was blocked by a PI3-K inhibitor and an anti-CD18 blocking mAb. Following the cross-linking of CCR5, monocytes synthesized high levels of M-CSF, RANTES, MIP-1{alpha}, and MIP-1ß associated with a readily detectable downmodulation of CD14, CD4, CCR5, and CXCR4 expression. The T312-enriched monocytes differentiated into dendritic cells (DCs) in the presence of interleukin-4 alone. After maturation with ß-interferon, the T312-induced DCs stimulated proliferation of allogeneic naïve CD4+ T cells accompanied by the synthesis of high levels of {gamma}-interferon in vitro. Furthermore, the T312-induced DCs were capable of stimulating antigen-specific human T- and B-cell immune responses in our hu-PBL-SCID mouse system. Finally, screening of other anti-chemokine receptor mAbs showed that select clones of mAbs against CXCR4 and CCR3 were also capable of facilitating enrichment of monocytes similar to T312. These results show that cross-linking of chemokine receptors on monocytes by appropriate mAbs leads to activation and differentiation of monocytes and that the method described herein provides an alternate simple strategy for adherence-based isolation of monocytes and generation of functional DCs.

Key Words: dendritic cell • monocyte • chemokine receptor • human immunodeficiency virus (HIV)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Peripheral blood monocytes are derived from bone marrow progenitor cells and are recruited to tissues, where they undergo differentiation into macrophages or myeloid dendritic cells (DCs) (1–4). Recruitment of monocytes from the recirculating pool into normal or inflamed tissues involves a series of cascading events, which include the generation of chemokine gradients and the expression of cell adhesion molecules and their cognate ligands (5). Ligation of the chemokine receptors activates ß1- and ß2-integrins (6), which in turn mediate adhesion of monocytes within tissues, where they differentiate into DCs. DCs are potent antigen-presenting cells (APCs) and have a central role in the activation and function of both innate and adaptive immune responses against infectious microorganisms (7). Dysfunction and potential loss of DCs have been associated with decreased antigen-specific T-cell responses and synthesis of lower levels of virus-suppressive Type-1 interferon (8–11).

Results from a number of studies have documented the enhanced potential of DCs to process and present antigen, and thus, DCs have been regarded as natural cellular "adjuvants." This functional attribute has led to clinical trials of DC-based immunotherapy not only in a number of animal tumor models and human malignancies (12) but also against a number of infectious disease agents. This view is highlighted by the recent finding that immunization of human immunodeficiency virus Type 1 (HIV-1)–infected patients with autologous DCs sensitized with chemically inactivated autologous HIV-1 led to a marked sustained decrease in viral load (13, 14). Essentially similar data, in terms of decreasing viral loads, were obtained using the simian immunodeficiency virus (SIV)–infected nonhuman primate model of human AIDS following immunization with autologous SIV-pulsed DCs (15); the results of this study were reasoned to be due to enhancement of both T-cell and neutralizing antibody responses. This finding was further supported using the hu-PBL-SCID model, in which human peripheral blood mononuclear cell (PBMC)–engrafted mice, following immunization with inactivated HIV-1–pulsed human DCs, were shown to generate high levels of HIV-1–specific T-cell and B-cell immune responses sufficient to protect these animals against challenge with virulent HIV-1 isolates (16, 17).

Human DCs for such studies are generally derived from culturing enriched populations of monocytes in vitro in media supplemented with varying combinations of cytokines, depending on the nature of the studies to be performed. Evidence has been accumulating in support of the notion that there exist subsets of DCs that differ in their expression of cell surface markers, in vivo trafficking patterns, and cytokines synthesized, which influences the quality of the T-cell response that is induced by such DCs (18). Thus, in vitro culture of monocytes with recombinant human granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) leads to their differentiation into myeloid DCs, and in vitro culture of those cultured in media containing GM-CSF and IL-3 leads to their differentiation into lymphoid DCs (19). There also appears to be evidence that a common precursor progenitor cell exists that can give rise to either myeloid or plasmacytoid DC subsequent to Flt-3 ligation (20). In the in vitro culture of the monocytes, GM-CSF functions as a survival and differentiation factor, whereas IL-4 induces differentiation of DCs by blocking their differentiation into the macrophage lineage (21, 22).

To study the biology of these DCs and to use them for in vivo studies, a large number of monocytes need to be isolated from the peripheral blood. This is accomplished using a variety of techniques, such as elutriation centrifugation (23), use of antibody-conjugated immunobeads (21, 24), and the more simple method of adherence of these cells to plastic (5, 24). Although the purity of the preparations using such procedures varies, it is clear that the former two techniques are expensive, requiring unique instrumentation and/or clinically trained staff, and labor intensive. Although the adherence-to-plastic method is simple, the yield of monocytes in such preparations varies among donors, and the results are difficult to reproduce.

We have previously reported that cross-linking of CXCR4 by a monoclonal antibody (mAb) that recognizes the extracellular loop 3 (ECL-3) region of CXCR4 induces homologous adhesion of T cells and enhances HIV-1 infection (25). Since fresh monocytes express a variety of chemokine receptors on their cell surface (26), we hypothesized that cross-linking of these chemokine receptors by an immobilized mAb might induce monocyte adhesion onto culture plates, providing a simple alternative procedure for the enrichment of monocytes from PBMCs.

Indeed, herein we present data that show that cross-linking the chemokine receptors CCR5, CXCR4, and CCR3 on monocytes by appropriate mAbs enhances adhesion of monocytes to plastic plates and that these adherent monocytes can be induced to differentiate along the macrophage or myeloid DC lineages with the use of distinct recombinant cytokines. This procedure will provide a relatively efficient and a more practical alternative for the isolation and study of monocytes and DC lineages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals.
SCID mice lacking functional T, B, and natural killer (NK) cells and BALB/c–rag2–/– common gamma–/– mice (27) were used in the present study. These mice were kept in the specific-pathogen-free animal facility of the Laboratory Animal Center at the University of the Ryukyus. The protocols for the care and use of the hu-PBL-SCID mice were approved by the Committee on Animal Research of the University of the Ryukyus before initiation of the present study.

Reagents.
RPMI-1640 medium was purchased from Sigma Chemical Co. (St. Louis, MO) and supplemented with 5% heat-inactivated fetal calf serum (FCS) (Sigma Chemical) (referred to as RPMI medium). Serum-free medium, AIM-V, was purchased from Life Technology (Grand Island, NY). Recombinant (r) human GM-CSF and IL-4 were produced in 293T cells transfected with pCMhGM-CSF and pCMhIL-4, respectively (RIKEN Gene Bank, Ibaraki, Japan), using the calcium phosphate method. The total protein and cytokine concentrations of the pooled culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and a functional assay, as described previously (16). rIL-2 was provided by the U.S. National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. rIFN-ß was purchased from Torey (Tokyo, Japan). rIFN-{gamma} and rM-CSF were purchased from Peprotec (London, UK). The PI3-K inhibitor LY294.002 LPS from Escherichia coli, OVA, keyhole limpet hemocyanin (KLH), and bovine serum albumin (BSA, fraction V) were all purchased from Sigma Chemical. Fluorescein isothiocyanate (FITC)-labeled E. coli, FITC-fibrinogen, FITC-gelatin, and FITC-collagen were purchased from Molecular Probes (Eugene, OR). Monocyte negative isolation kits were purchased from Dynal (Oslo, Norway). Cell proliferation kits were purchased from Roche Diagnostics (Mannheim, Germany). ELISA kits for human IL-1ß, IL-4, IL-6, IL-10, IL-12 p70, tumor necrosis factor (TNF)-{alpha}, M-CSF, MIP-1{alpha}, MIP-1ß, and RANTES were purchased from Biosource (Camarillo, CA). ELISA kits for {gamma}-interferon (IFN–{gamma}) were purchased from R&D Systems, Inc. (Minneapolis, MN). Naïve CD4+ T-cell isolation kit was purchased from MACS (Gladbach, Germany).

The mAbs produced in our laboratory included two rat anti-human CCR5 N-terminus clones (clone T312 immunoglobulin [Ig]G1, clone T227 IgG2b); three rat anti-human CXCR4 (clone A145 IgG1, clone A120 IgG2b, clone A80 IgG1) (25); rat anti-human T-cell leukemia virus (HTLV)-I (clone LAT-27, IgG2b) (28); rat anti-hepatitis C virus (HCV) (clone Mo-8, IgG2b) (29); rat anti-human OX40 (clone W4–54, IgG2b, Tanaka et al., unpublished data), and mouse anti-human OX40L (clone 5A8, IgG1) (30). The other mAbs used included mouse IgG anti-human CD4, SIM-2, and SIM-4 (obtained from the U.S. NIH AIDS Research and Reference Reagent Program), the clones OKT-4, OKT-8, and 60-bca anti-CD14 (obtained from ATCC, Rockville, MD). These mAbs were purified from SCID mouse ascites fluids by Superdex G-200 gel filtration (Amersham Bioscience, Uppsala, Sweden). Commercially available mAbs used were mouse IgG anti-human CD4, CD11c, CD80, CD83, CD86, HLA-DR (Coulter Inc., Hialeah, FL), and mouse IgG anti-human CD11a, CD11b, CD14, CD18, CD29, CD51, and CD61 (BD Pharmingen, San Diego, CA). Additional anti-human chemokine receptor mAbs, such as anti-CCR1 (mouse IgG2b, Cat #MAB145), CCR2 (mouse IgG2b, Cat #MAB150), CCR3 (rat IgG2a, Cat #MAB155), CCR5 (mouse IgG2b, Cat #MAB180), and CCR8 (rat IgG2b, Cat #MAB1429), were purchased from R&D Systems, Inc. (Minneapolis, MN); and the mAbs against CXCR4 (clone 12G5, mouse IgG2a) and CCR5 (clone 2D7, mouse IgG2a) were purchased from BD Pharmingen.

Cultivation of Monocytes.
PBMCs were isolated from heparinized (5 U/ml) blood of normal healthy donors by a standard density gradient centrifugation at 400 g using lymphocyte separation medium (Sigma Chemical) for 15 mins at room temperature. The cells at the interface were collected and washed three times in cold phosphate-buffered saline (PBS) containing 0.1% BSA (BSA-PBS). PBMCs were resuspended in RPMI medium or serum-free AIM-V medium at 5 x 106 cells/ml. Then, 1 ml of the cell suspension was dispensed into individual wells of 12-well plates (BD Pharmingen), which were precoated with various mAbs (5 µg/ml) for 1 hr at 37°C. PBMCs were allowed to adhere 2 hrs or overnight at 37°C in a 5% CO2 humidified incubator. Nonadherent cells were removed by gentle washing three times in BSA-PBS. The remaining adherent cells were then cultured in RPMI medium. For some experiments, monocytes were purified using a monocyte negative isolation kit and were used at 1–2 x 105 cells/ml. For the generation of macrophages, adherent or immunomagnetic bead-enriched CD14+ monocytes were cultured in the presence of 20 ng/ml M-CSF for 6 days in a 5% CO2 humidified incubator. For the generation of DCs, the media was supplemented with either GM-CSF (500 ng/ml) and IL-4 (200 ng/ml) or IL-4 (25 ng/ml) alone. Immature DCs were obtained after 5–6 days of culture. For maturation, the immature DCs were cultured in the presence of human ß-interferon (IFN-ß) (1000 U/ml) for an additional day. For sensitization with antigens, the immature DCs at Day 6 were cultured in the presence of either 100 µg/ml OVA or KLH for 1 day and then matured with IFN-ß for an additional day. Viable cell number was assessed on an aliquot of such cells using staining with 0.1% eosin-Y.

Flow Cytometry.
Phagocytosis and cell surface markers were determined using fluorescence activated cell sorter (FACS) Calibur and Cell Quest software (BD Pharmingen). Cell samples were Fc-blocked by incubation in media containing 2 mg/ml of human IgG in PBS containing 0.1% NaN3 and 2% FCS (FACS buffer) on ice for 15 mins and were then stained with appropriate fluorescent dye–conjugated reagents on ice for 30 mins, according to the manufacturer’s instructions. After washing with FACS buffer, cells were fixed in 1% paraformaldehyde-containing FACS buffer and analyzed. For quantitation of phagocytosis, sample cells (1 x 106) in 0.2 ml of RPMI medium were cultured in the presence of FITC-labeled E. coli at a cell to bacterium ratio of 1:10 to 1:100 for 1 hr at 37°C in a 5% CO2 humidified incubator. After incubation, these cells were washed once in FACS buffer, fixed with 1% PFA, and then analyzed.

There were significant individual variations in the percentage of CCR5-positive cells in PBMCs. The percentages of T312-positive cells were as follows: 6%–14% of CD14+ monocytes, 11%–43% of CD3+ T cells, 6%–15% of CD3+ CD4+ T cells, and 5%–14% of CD20+ B cells. Overnight incubation of PBMCs in medium alone led to a marked increase in the frequency of monocytes that expressed CCR5 (up to 70%), as detected using the T312 mAb. However, there was no detectable change of CCR5 expression in the other cell subsets (data not shown).

DC Functional Assays.
Several assays were employed to determine whether the cell population that was being cultured belonged to the DC lineage. This included quantitation of the levels of IL-12 p70 and IL-10 produced in the culture supernatants of the potential DCs, as determined by ELISA; ability of the cells to induce allo-proliferation; and a unique in vivo assay. For the assessment of allo-proliferative potential, the mature or immature DCs to be tested were cultured at 1 x 105 cells/ml in RPMI medium in the presence of LPS (1 µg/ml) and IFN-{gamma} (100 ng/ml) for 24 hrs. The ability to induce allogeneic stimulation was determined by co-culturing naïve CD4+ T cells (1 x 105) with these potential DCs (0.5 x104) in triplicate in a 96-well plate in a final volume of 0.2 ml in RPMI medium containing 20 U/ml IL-2 for 7 days. The level of proliferation of the CD4+ T cells was assessed by the bromodeoxyuridine (BrdU)–incorporation ELISA method (31).

The in vivo function of DCs was determined using the hu-PBL-SCID mouse system, as previously described (16). Briefly, a SCID mouse received antigen-pulsed mature DCs (5 x105 cells) with autologous fresh PBMCs (3 x106 cells) in a final volume of 0.1 ml in RPMI medium injected directly into the spleen, followed 7 days later with the ip injection of the same number and source of DCs pulsed with the same antigen. Five days later, the mice were sacrificed, and blood was collected by cardiocentesis and human lymphocytes were recovered from both the peritoneal cavity and the spleen. The immune sera were assayed for human antibody titers against OVA or KLH by ELISA using peroxidase-labeled goat anti-human IgG, as described previously (16). For the measurement of antigen-specific human T-cell immune responses, the human lymphocytes (2 x 106 cells) collected from the immunized mice were cultured for 2 days at 37°C in a 5% CO2 humidified incubator with 2 x105 autologous APCs (adherent PBMCs) in the presence or absence of either 5 µg/ml OVA or KLH in a volume of 0.5 ml in individual wells of a 24-well microtiter plate (BD Pharmingen). Media used consisted of RPMI medium supplemented with 20 U/ml of IL-2. The concentration of human IFN-{gamma} produced in the culture supernatants was determined by standard ELISA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cross-Linked CCR5 Induces Strong Adhesion of Monocytes.
Twelve-well microtiter plates were first coated with the rat anti-human CCR5 N-terminus antibody (IgG1, clone T312) or an isotype control mAb. Then, PBMCs (5 x 106/ml) isolated from several normal donors were individually incubated overnight at 37°C in a 5% CO2 humidified incubator in a volume of 1 ml of RPMI medium in each well of the plate. Following incubation, the nonadherent cells were removed. As shown in Figure 1aGo, there appeared to be a marked increase in the number of cells that remained adherent to the wells precoated with the T312 mAb as compared with the control wells. As seen in Figure 1bGo, of interest was the finding that the nonadherent cells from the T312 mAb-coated wells were selectively depleted of CD14+ cells (0.5%), compared with the nonadherent cells from the control wells (8%). The input unfractionated PBMCs from the various donors used for this study contained levels of CD14+ cells that were essentially similar to the levels noted in the control wells, indicating that incubation of the PBMCs in the T312 precoated wells led to the select adhesion of the CD14+ cells. Lymphoid cells other than CD14+ monocytes were also present in the population of cells that adhered to the T312 mAb-coated wells, which mainly consisted of T and B cells, constituting up to 20% of the total adherent cells (data not shown). It is important to note that the addition of the T312 mAb to aliquots of the PBMCs from the same donors before incubation in the microtiter wells or the addition of the same T312 mAb to the PBMCs following dispensing of the cells into the microtiter wells did not lead to increased adhesion of the CD14+monocytes, providing evidence indicating that the antibody had to be cross-linked for the adhesion to occur. A study of the kinetics of cell adhesion to the T312 mAb-coated wells was also performed. It should also be noted that there was a difference in the incubation time for maximal yield of monocytes following incubation of PBMCs in wells precoated with the T312 mAb, compared with the control antibody. Thus, where a 2-hr incubation was sufficient to obtain the maximum yield of monocytes by incubation of PBMCs in the control antibody-coated wells, it required an overnight or 24-hr incubation in the T312 mAb-coated wells (data not shown). Thus, for all further studies, adherent cells were isolated by incubation of the PBMCs either for 2 hrs in control wells or overnight in T312 mAb-coated wells, unless otherwise noted.


Figure 1
View larger version (55K):
[in this window]
[in a new window]
  		Figure 1. Stable adhesion of monocytes onto the bottom of plastic wells precoated with an anti-human CCR5 mAb. Aliquots (1 ml) of PBMCs at 2 x 106/ml of 5% FCS-RPMI medium were cultured overnight in individual wells of a 12-well plate that had been previously coated either with clone T312 rat IgG1 anti-human CCR5 or an isotype control mAb. (a) The morphology of the adherent cells as seen by phase-contrast microscopy at x100. (b) Nonadherent PBMCs from each well were collected and analyzed by flow cytometry. The percentages of CD14+ monocytes in the lymphocyte and monocyte gate in nonadherent PBMCs are noted in the double-staining profile with anti-CD3 and anti-CD14 mAbs. FS, forward scatter; SS, side scatter.

 
Since binding of CCR5 by its natural ligands is known to induce intracellular signaling via the PI3-K pathway, experiments were carried out to determine if such signaling was a prerequisite for the stable binding of the monocytes to the T312 mAb-coated wells. The above experiment was repeated, with the only difference being that an aliquot of each of the donor PBMCs was incubated in the T312 mAb-coated wells in the presence of 5 µM of the PI3-K inhibitor LY294.002. Representative data shown in Figure 2aGo demonstrate that the addition of the PI3-K inhibitor completely blocked the cell adhesion. Flow cytometric analysis of the nonadherent cells in the wells containing the PI3-K inhibitor confirmed the presence of a high frequency of CD14+ cells, denoting the lack of adhesion of the CD14-expressing cells (data not shown). It was also of interest that the addition of various concentrations of soluble or immobilized RANTES, MIP-1{alpha}, and MIP-1ß did not enhance monocyte adhesion (data not shown), denoting that simple ligation of the chemokine receptors was not sufficient to facilitate adhesion.


Figure 2
View larger version (76K):
[in this window]
[in a new window]
  		Figure 2. Microscopic analysis of the mechanisms for monocyte adhesion following CCR5 cross-linking. (a) PBMCs were incubated for 2 hrs in control mAb-coated wells (control/med) or overnight in T312 mAb-coated wells in medium alone (T312/med) or in the presence or of 5 µM PI3-K inhibitor, LY294.002 (T312/LY294). (b) PBMCs were cultivated overnight in T312 mAb-coated wells in medium alone (control) or in the presence of 10 µg/ml of each mAb: anti-CD18, CD29, or CD11a. In these experiments, nonadherent cells were removed by gentle washing, and the wells were analyzed microscopically at x200.

 
It has been previously documented that adhesion of monocytes onto plastic plates is mediated by fibrinogen binding by the ß2-integrins Mac-1 (CD11b/CD18) and/or p150/95 (CD11c/CD18) (32, 33). As shown in Figure 2bGo, the T312 mAb-induced adhesion of monocytes was blocked by anti-CD18 blocking mAb (with specificity for the common chain of ß2-integrins), but not anti-CD29 mAb (ß1-integrin) or anti-CD11a (another component of the ß2-integrin, LFA-1), indicating an involvement of the ß2-integrins in the enhanced adhesion of monocytes by T312 mAb-mediated CCR5 cross-linking.

Cytokine Profile and Phenotype Analysis of Monocytes Adhered to the T312 mAb-Precoated Wells.
In an effort to define the cytokine profile characteristic of the monocyte population, it was reasoned that depletion of nonmonocyte lymphoid cells before analysis would be important. The magnetic bead negative selection technique for CD14+ cells was thus used to deplete such nonmonocyte lymphoid cells, and the subsequent enriched monocytes were then incubated overnight in wells precoated with either T312 or control mAb, and the supernatant fluids were collected. Representative data from three experiments are shown in Figure 3aGo. As seen, the T312 mAb-stimulated monocytes secreted significant levels of M-CSF, MIP-1{alpha}, MIP-1ß, and RANTES. The CD14+ monocytes are likely to be the major cell lineage that responded to the T312 mAb stimulation by the production of cytokines, because PBMCs depleted of CD14+ monocytes failed to synthesize detectable levels of the same cytokines. An aliquot of the same supernatants was also screened for levels of GM-CSF, TNF-{alpha}, IL-1ß, IL-12, or IL-10, but each was found to be below 0.1 ng/ml. A variety of rat and mouse mAbs that had been generated in our laboratory with specificity for HTLV-1, HIV-1, HCV, OX40L, OX40, or IL-2R were also used to precoat the wells and were screened for their ability to induce cytokine synthesis by purified monocytes in parallel with the use of T312 mAb. As expected, only monocytes incubated in the wells precoated with T312 mAb produced M-CSF, MIP-1{alpha}, MIP-1ß, and RANTES, denoting an element of specificity for the T312 mAb (data not shown).


Figure 3
View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Cytokine profile and phenotypic analysis of purified monocytes following CCR5 cross-linking. (a) CD14 negatively selected monocytes at 2 x 105/ml in RPMI medium in T312 mAb-coated (T312-treated) or control wells (control) were cultured overnight, and cytokines produced in the culture supernatants were assayed by ELISA. Values of <0.1 ng/ml were below the sensitivity of the assay employed. (b) Aliquots of monocytes that were cultured in control wells (thick dark lines) or those cultured in T312 mAb-coated wells (thin dark lines) were analyzed for the expression of CD14, CD4, CCR5, and CXCR4 by routine flow cytometry. The broken lines show background staining of cells with isotype control mAbs.

 
Phenotypic analysis of monocytes adhered to the T312 mAb-precoated wells consistently showed a marked down-regulation of CD14, CD4, CCR5, and CXCR4 (a representative profile is shown in Figure 3bGo). Downmodulation of the CD14 on the cell surface was not attributed to the shedding of CD14, since there was no difference in the level of soluble CD14 between the supernatant fluids from the wells precoated with the T312 mAb and those from the control wells, as determined by ELISA (data not shown).

CCR5 Cross-Linking Stimulates Monocyte Differentiation into DCs.
To test whether the T312 mAb-induced CCR5 cross-linking has the potential to differentiate monocytes into functional DCs, aliquots of PBMCs were first incubated either in the T312 mAb-coated wells overnight or the control mAb-coated wells for 2 hrs (for the generation of conventional DCs), the nonadherent cells were removed, and then the adherent cells were further cultured in the presence or absence of either GM-CSF and IL-4 or IL-4 alone for 5 or 6 days, respectively.

As shown in Figure 4aGo, the adherent cells incubated in T312-coated wells appeared to have a DC type of morphology, which was confirmed by flow cytometric analysis (see Fig. 4bGo). Interestingly, the T312 mAb-stimulated monocytes, when cultured in the presence of IL-4, regardless of the presence or absence of GM-CSF, exhibited higher mean density levels of CD86 and lower levels of CD1a than did control conventional DCs that were cultured in media containing both GM-CSF and IL-4. The DCs cultured in the presence of GM-CSF and IL-4 in the T312 mAb-coated wells had a phenotype that was basically similar to that of the ones cultured in IL-4 alone, and these are both representative profiles of immature DCs. Kinetic studies of the differentiation of these T312 mAb-stimulated immature DCs showed that the optimal level of maturation was achieved by cultivation in the presence of IFN-ß for an additional day.


Figure 4
View larger version (57K):
[in this window]
[in a new window]
  		Figure 4. Morphological and flow cytometric analyses of adherent PBMCs following maturation in different culture conditions in vitro. (a) PBMCs at 5 x106/ml in RPMI medium were cultured overnight in wells precoated with T312 mAb. After removal of nonadherent cells, adherent cells were cultured in RPMI medium (medium) or in the presence of 500 ng/ml GM-CSF and 200 ng/ml IL-4 (GM-CSF+IL-4) or 25 ng/ml IL-4 alone (IL-4) for 5 days. Morphology was observed microscopically at x200. (b) The T312-induced immature DCs were generated from PBMCs adhered to the T312 mAb-coated wells during overnight incubation followed by cultivation in the presence of either GM-CSF and IL-4 (T312-DC in GM+IL-4) or IL-4 alone (T312-DC in IL-4). For comparison, control conventional DCs were generated from 2 hr–adherent PBMCs in control wells followed by cultivation in GM-CSF and IL-4 for 6 days (cont. DC). After maturation for an additional day with IFN-ß, aliquots of these cells were subjected to flow cytometric analysis, and the profiles for the expression of HLA-DR, CD86, CD83, CD14, CD11c, and CD1a are shown.

 
We next sought to examine the relative phagocytic activity of monocyte-derived DCs or macrophages that were cultured in various conditions. As seen in Figure 5Go, macrophages obtained following differentiation of a highly enriched population of CD14+ monocytes in M-CSF–containing media in general gave a higher relative phagocytic index than did DCs generated in media containing GM-CSF and IL-4. In addition, those that were stimulated with T312 mAb and cultured in media containing either GM-CSF and IL-4 or IL-4 alone were less phagocytic than the control macrophages generated by M-CSF. Interestingly, the T312 mAb-stimulated monocytes, following incubation with media containing M-CSF alone, gave slightly lower phagocytic values than the normal macrophages cultured in the same media. These data seem to indicate that cross-linking of CCR5 influences monocyte differentiation into DC rather than macrophage, even in the presence of M-CSF.


Figure 5
View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Phagocytic activities of cultured macrophages or immature DCs. Various DCs were generated as described in the legend for Figure 4Go, and macrophages were generated from 2 hr–adherent PBMCs followed by cultivation in the presence of M-CSF for 6 days. These sample cells in RPMI medium were incubated with a 10-fold excess of FITC-labeled E. coli particles for 1 hr at 37°C. After washing, the levels of uptake of FITC-labeled E. coli by cells were determined by flow cytometry with gating for macrophages and DCs using standard forward and side scatter.

 
We further determined the relative levels of viability of the cells that adhered either to the T312-coated wells overnight or to control wells for 2 hrs followed by cultivation in medium alone or media containing either GM-CSF and IL-4 or IL-4 alone for 5 or 6 days, respectively. Figure 6aGo shows, first of all, that whereas there was variation in the number of viable cells recovered among donors of the PBMCs, the yields of viable cells generated in the T312 mAb-coated or control wells followed by cultivation in GM-CSF and IL-4 were basically similar. In contrast, aliquots of the same cells incubated in T312 mAb-coated or control wells gave different yields following incubation with IL-4 alone. The cells stimulated by T312 mAb gave significantly higher viable yields than those incubated in control wells. The overnight adherent monocytes could differentiate into functional DCs in the presence of GM-CSF and IL-4, but the cell yield was lower than those obtained from the 2-hr adherent cells (data not shown).


Figure 6
View larger version (64K):
[in this window]
[in a new window]
  		Figure 6. Yields of DCs and IL-12 production. (a) Immature DCs were generated by either incubation of aliquots of PBMCs from three different donors in the control wells (control) or in the T312 mAb-coated wells (T312-treated) and then for 6 or 5 days, respectively, with GM-CSF and IL-4 or IL-4 alone, as described in the legend for Figure 4Go. Numbers of viable cells that were not stained by eosin-Y were manually counted using a hemocytometer. The means from triplicate determinations are shown. (b) DCs from a donor before (immature) or after IFN-ß treatment (mature) were stimulated by 1 µg/ml LPS and 100 ng/ml IFN-{gamma} for 24 hrs. IL-12 p70 levels in the culture supernatants were determined by ELISA.

 
Aliquots of these in vitro–generated immature DCs were subsequently challenged with LPS and IFN-{gamma} for 48 hrs, and supernatant fluids were assayed for levels of IL-12 p70. Controls consisted of aliquots of the conventional DCs obtained from the same PBMCs in wells coated with control IgG1 instead of T312 mAb. As seen in Figure 6bGo, there were significant levels of IL-12 synthesized by both these populations of immature DCs, and the importance of the levels appeared quite similar. Furthermore, the IL-12–producing activity of these immature DCs was lost after full maturation by incubation with IFN-ß.

Function of DCs.
In an effort to determine whether the T312 mAb-induced DCs were as competent as conventional DCs, these immature DC populations were matured by cultivation in media containing IFN-ß for 1 day and the aliquots dispensed into microtiter wells and standard mixed lymphocyte reaction (MLR) studies performed. The T312 mAb-induced DCs (termed T312/IL-4 or T312/GM-IL-4) and the conventional DCs (termed control/GM-IL-4) were co-cultured with allogeneic naïve CD4+ T cells. Controls consisted of CD4+ T cells cultured alone or DCs cultured alone. The cultures were performed at a DC to T-cell ratio of 1 to 20, and each combination was cultured in triplicate for 7 days and proliferation assessed using BrdU incorporation by the ELISA method. Supernatant fluids from the MLR cultures were also assayed for levels of IFN-{gamma}, IL-4, and IL-10. As shown in Figure 7aGo, there was no significant difference in the proliferation-inducing activity among the three preparations of DCs. However, an obvious difference was seen in the levels of IFN-{gamma} synthesized from the naïve CD4+ T cells (Fig. 7bGo). The T312 mAb-induced DCs generated in the presence of either GM-CSF and IL-4 or IL-4 alone stimulated higher production of IFN-{gamma} than the control DCs. None of these DCs induced production of IL-4 or IL-10 from the CD4+ T cells by co-culture. The T312 mAb-induced DCs did not produce detectable levels of human IFN-{gamma}, even following stimulation with LPS or CD40L (data not shown), indicating that the allogeneic naïve CD4+ T cells are the likely source of IFN-{gamma} following co-culture and stimulation with DCs. These results indicate that the T312 mAb-induced DCs acquired a relatively higher Th1-polarizing capacity than control conventional DCs.


Figure 7
View larger version (47K):
[in this window]
[in a new window]
  		Figure 7. T312 mAb-induced DCs stimulate allogeneic naïve CD4+ T cells to proliferate and produce IFN-{gamma}. Allogeneic naïve CD4+ T cells were co-cultured with IFN-ß–matured DCs that had been generated from the adherent PBMCs, as described in the legend for Figure 4Go in RPMI medium supplemented with 20 U/ml IL-2 for 7 days. (a) Cell proliferation was determined by BrdU incorporation. Mean values from six wells and standard errors are shown. Controls consisted of culturing either DCs or T cells alone. (b) IFN-{gamma} production in the culture supernatants was determined by ELISA.

 
Furthermore, we tested and compared the ability of these DCs to induce human antigen-specific T- and B-cell immune responses in vivo. For this purpose, we used our hu-PBL-SCID mouse model (16). SCID mice were engrafted with normal PBMCs together with antigen (OVA)–pulsed autologous T312 mAb-induced or conventional mature DCs. A booster injection with antigen-pulsed homologous source of mature DCs was performed after 7 days. Immune sera and spleen cells were harvested after 5 days following the booster injection. Figure 8Go shows that immunization with the T312 mAb-induced and OVA-pulsed DCs was more efficient in inducing antigen-specific T-cell immune responses in the hu-PBL-SCID mice, as determined and defined by human IFN-{gamma} production by the immune cells in vitro, than the conventional DCs pulsed with OVA. IL-4 was not produced from those immune cells (data not shown). Human OVA-specific IgG were also induced by both types of the conventional and T312 mAb-induced DCs up to a titer of 1:400, as determined by ELISA (data not shown). Essentially similar results were obtained when mice were immunized with another antigen, KLH (data not shown). These data indicate that the T312 mAb-induced DCs, in general, appear to be relatively more efficient than conventionally derived DCs in inducing antigen-specific IFN-{gamma} responses in vivo.


Figure 8
View larger version (31K):
[in this window]
[in a new window]
  		Figure 8. T312 mAb-induced DCs trigger human antigen–specific T-cell responses in hu-PBL-SCID mice. DCs were generated as described in the legend for Figure 4Go, followed by exposure to antigen (100 µg/ml OVA or KLH) for 24 hrs and by maturation with IFN-ß for an additional 24 hrs. These DCs (5 x 105 cells) together with autologous fresh PBMCs (3 x 106 cells) were mixed in 0.1 ml RPMI medium and transplanted into the spleen of SCID mice. Booster immunization was made by injection of the same numbers of antigen-loaded DCs (ip) on Day 7. After 5 days, cells obtained from the spleen and peritoneal cavity of the mice were cultured with autologous APCs (adherent PBMCs during 2-hr incubation in normal wells) in the presence (APC +Ag) or absence (APC alone) of 5 µg/ml OVA for 2 days, and the supernatants were assayed for human IFN-{gamma} by ELISA. Values depicted reflect the mean levels of IFN-{gamma} of triplicate cultures (SD was <10%).

 
Effect of Cross-Linking by Other mAbs Against Chemokine Receptors.
Based on the results from the above studies, additional experiments were carried out to examine the specificity of the ligation of the CCR5 coreceptor by the T312 mAb. Thus, a battery of mAbs against human chemokine receptors expressed by monocytes, including CCR1, CCR2, CCR3, CCR5, CCR8, and CXCR4, were used to precoat microtiter wells, and aliquots of purified monocytes were incubated in such antibody-coated wells. The level of M-CSF synthesized by the cells following overnight incubation was used as an index of monocyte stimulation. As seen in Figure 9Go among the anti-human CCR5 mAbs tested, Mab180 (mouse IgG anti-CCR5 N-terminus), but not T227 (rat IgG2a anti-CCR5 N-terminus) or 2D7 (mouse IgG anti-CCR5 ECL-2), was as effective as T312 mAb in the stimulation of monocytes. Among the anti-human CXCR4 mAbs, clone A120 (rat IgG2b anti-CXCR4 ECL1&2) and A80 (rat IgG1 anti-CXCR4 ECL-3) were also capable of stimulating monocytes. Another monocyte-stimulating mAb was the rat IgG2a anti-CCR3 mAb. These mAbs that successfully stimulated monocytes also promoted monocyte adhesion, and A120 anti-CXCR4 mAb in particular was very potent in inducing adhesion and differentiation of monocytes similar to the data derived using the T312 mAb (data not shown). Whether subtle differences exist in the adherent cells enriched by the mAbs that were active in such an assay remains to be determined.


Figure 9
View larger version (60K):
[in this window]
[in a new window]
  		Figure 9. Screening of the other anti-chemokine receptor mAbs that can stimulate monocytes. Purified monocytes at 1 x 105/ml in RPMI medium were cultured in wells precoated with various mAbs overnight, and the culture supernatants were assayed for M-CSF by ELISA. The values depicted represent the mean of triplicate cultures with SD of <10%.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we present data showing that cross-linking of CCR5 as well as select other chemokine receptors (CXCR4 and CCR3) by mAbs immobilized onto plastic microtiter wells leads to enhanced adhesion of monocytes followed by synthesis of M-CSF and a set of ß-chemokines and that incubation of these adherent cells with GM-CSF and IL-4 or IL-4 alone leads to their differentiation along the DC lineage. This functional activity observed following chemokine receptor cross-linking is in contrast to the data obtained with FcR cross-linking (34). This enhanced adherence of monocytes apparently improves the efficacy of adhesion-based monocyte enrichment from fresh PBMCs.

One contributing mechanism that facilitates such tight adhesion may involve the enhanced affinity of the ß2-integrins Mac-1 and/or p150/95, which bind to fibrinogen, an extracellular matrix protein, since such adhesion was markedly inhibited in the presence of mAb anti-CD18 (a common chain of the ß2-integrin) but not anti-CD11a (a component of another ß2-integrin, LFA-1). This view is supported by data obtained by flow cytometric analysis of the T312 mAb-stimulated monocytes, compared to those incubated in control mAb-coated wells. Thus, the T312 mAb-stimulated cells but not the control cells showed a markedly increased density of staining with FITC-fibrinogen, but not with FITC-gelatin or collagen (data not shown). Although sensitization of chemokine receptors by their respective cognate chemokine ligands is known to activate ß1- and ß2-integrins (5), it is important to note that the addition or precoating of wells with chemokines that bind to CCR5 (i.e., RANTES, MIP-1{alpha}, and MIP-1ß), respectively, did not induce such enhanced monocyte adhesion in vitro in the present study (data not shown). Thus, it is likely that antibody-mediated cross-linking of chemokine receptors initiates an enhanced degree of activation of monocytes, probably through an extraordinary degree of polarization of the receptors onto the plates. This is supported by the observations made by Lee et al. (35) that the mode of monocyte activation by cross-linking of chemokine receptors by gp120 of HIV-1 particles was stronger than that induced by a simple interaction between CCR5 and CXCR4 with their respective chemokines. The inhibition of the T312 mAb-induced monocyte adhesion by the PI3-K-specific inhibitor LY294.002 indicates that the T312 mAb-mediated CCR5 cross-linking also stimulates the G-protein signal cascade, as documented previously for gp120 of HIV-1 (35) and chemokines (36). Since PI3-K activation by chemokines also induces polarization of adhesion molecules and facilitates migration (5, 37), it is likely that it is either the mere strength of the signal that is induced by cross-linking of CCR5 by T312 mAb or the potential pulling together of additional, as-yet-unknown proteins that distinguishes between enhanced adhesion and mere signaling.

It is worth noting that the tight adhesion was not induced by another rat IgG anti-CCR5 mAb, termed clone T227. The difference between anti-CCR5 clone T312 and T227 is that the T227 mAb recognizes a region located within the N-terminus of the CCR5 molecule that has been further localized to a peptide spanning the CCR5 N-terminus amino acids 1–20 but not 2–21, whereas the clone T312 mAb recognizes both of the peptides, with an affinity similar to the clone T227 mAb (Tanaka et al., unpublished data). These two mAbs compete with each other in binding to CCR5. Thus, these results indicate that activation of monocyte by CCR5 cross-linking is epitope-dependent rather than antibody affinity-dependent. This view is further supported by the observation that the use of yet another immobilized CCR5-specific clone, ECL-2 mAb, did not lead to enhanced adhesion of monocytes, indicating a requirement for epitope specificity to facilitate adhesion. Similarly, with regard to CXCR4, the clones A145 mAb (anti–N-terminus) and 12G5 (anti-ECL1&2) did not induce significant monocyte adhesion, in contrast to A120 mAb (anti-ECL1&2) and A80 mAb (anti–ECL-3). The clone A120 mAb blocks the binding of SDF-1 (the natural ligand for CXCR4) and inhibits infection by the CXCR4-tropic HIV-1 strains, whereas the clone A80 mAb does not block SDF-1 binding but stimulates homologous T-cell aggregation and thus enhances infection of the CXCR4 and CCR5-tropic HIV-1 strains (25). However, it is still unclear which regions (epitopes) of each chemokine receptor are the hot spots for the induction of strong activation signals on monocytes, and this topic thus requires further study.

Along with the enhanced adhesion facilitated by cross-linking of the chemokine receptor, such cross-linking also induced downmodulation of cell surface expression of CD14, CD4, CCR5, and CXCR4 and cytokine production by monocytes. It is of interest that similar effects on monocytes have been induced by prokineticin 1 (PK1)/endocrine gland-derived vascular endothelial growth factor (EG-VEGF), and its G protein-coupled receptors (PKR1 and PKR2) (38). Downregulation of CD14 was reported to be induced by IL-4 or IL-13 at the transcriptional level (39) or by shedding, which was induced by antibody-mediated cross-linking of CD14 or stimulation by LPS and IFN-{gamma} (40). However, these are not the likely mechanisms in studies reported herein, since there was little if any production of IL-4 and IL-13 and no detectable levels of soluble CD14 in the culture supernatants. It has been shown that downmodulation of cell surface expression of CD14, CD4, and CXCR4, but not CCR5 on monocytes, is induced by PKC activation by phorbol 12-myristate 13-acetate (PMA) (41, 42) and that CCR5 downmodulation is induced by CCR5-binding chemokines at only high concentrations as a result of CCR5 internalization (43). Whether these pathways are activated by cross-linking of the chemokine receptors is currently not known. It has also been shown that there is a molecular association between CD4 and the chemokine receptors or among the various chemokine receptors (44, 45). Thus, it can be speculated that CCR5 or CXCR4 cross-linking may induce homo- or hetero-oligomerization of the cell surface CD14, CD4, CXCR4, and/or CCR5, and is then internalized.

In the early studies on monocyte adhesion (5) it was shown that adherence onto plastic plates stimulates monocytes to activate M-CSF, IL-1ß, or TNF-{alpha} gene expression and that LPS stimulation further enhances the synthesis of these cytokines. Similarly, activation of monocytes via the PK1 pathway following adhesion requires additional stimulation by LPS to promote the production of IL-12 p70 and TNF-{alpha} (46). Thus, it is clear that the mechanisms for cytokine production in the studies described herein likely involve distinct pathways other than those discussed above. In addition, the addition of soluble chemokines or the use of immobilized chemokines with specificity for CCR5 and CXCR4 did not induce cytokine production in the present conditions (data not shown). Differences in the precise pathways that are triggered in the studies reported herein are a subject of current study.

The observation that not only CCR5 but also CXCR4 and CCR3 cross-linking triggered production of M-CSF by monocytes and induced differentiation in the absence of exogenously added M-CSF or GM-CSF was unexpected. For differentiation of monocytes into macrophages in vitro in the absence of these cytokines, extracellular matrix proteins are known to have profound influence (47). It has been previously reported that human monocytes differentiated into CD14-low macrophages in a fibronectin-containing medium without the addition of exogenous cytokines (48). Thus, bovine fibronectin in our RPMI medium may have some additive effects on chemokine receptor–stimulated monocyte differentiation, although the use of serum-free medium for monocyte cultivation in T312 mAb-coated wells did not influence the present results (data not shown). Although cross-linking of CCR5, CXCR4, or CCR3 resulted in M-CSF production from monocytes, anti-M-CSF– or GM-CSF–neutralizing mAbs did not block monocyte differentiation into macrophages (data not shown). Thus, we assume that chemokine receptor cross-linking may bypass the requirement of M-CSF stimulation. Since the ligation of M-CSF receptor (CD115) stimulates PI3-K (49, 50), the PI3-K activation as a result of the chemokine receptor cross-linking may be responsible for the de novo monocyte differentiation into macrophages. The mechanisms by which cross-linking of chemokine receptors on monocytes induced the differentiation of these cells into DCs without the addition of exogenous GM-CSF in the presence of IL-4 are also under current study. The cross-linking may function as survival and differentiation factor for the generation of DCs from monocytes, because monocytes in the absence of chemokine receptor cross-linking failed to survive when cultured in media containing IL-4 alone.

The primary objective of the present study was to find an alternate, more-efficient and practical method for the in vitro generation of functional DCs with potent Th1-inducing capacity in vivo. Various cytokine cocktails have been applied for generation of functional DCs, including combinations of IL-3 and IFN-ß, Flt3L and IL-4, CD40L alone, or GM-CSF, IL-4, and the proinflammatory cytokines GM-CSF and Type I IFN (51–56). In addition, it has been reported that the methods of monocyte isolation, such as adherence or CD14+enrichment by MACS (which results in CD14 cross-linking), influence cytokine production from generated DCs (i.e., adherent monocyte-derived DCs were more potent in producing IL-12, IL-10, and TNF-{alpha} and in stimulating Tc1 than those from CD14 positively selected monocytes) (24). Because the DCs generated in the studies reported herein by CCR5 (or CXCR4 or CCR3) cross-linking in the presence of either GM-CSF and IL-4 or IL-4 alone were relatively more potent than conventional DCs in stimulating IFN-{gamma} production from allogeneic naïve CD4+T cells in vitro and OVA- or KLH-specific T cells in vivo, it is indicated that in studies that require Th1-polarizing DCs, the cross-linking of CCR5, CXCR4, or CCR3 on monocytes should be considered. The higher density of expression of CD86 on the chemokine receptor cross-linked DCs may have some influence for Th1-inducing capacity, although the mechanism at present still remains unknown.

Based on the results of the present study, we suggest that the use of cross-linking of CCR5, CXCR4, or other chemokine receptors for the generation of functional DCs ex vivo provides an alternate and simple tool that will facilitate the therapeutic use of DCs. Thus, studies of DC-based immunotherapy in patients with HIV-1 infection that have been reasoned to require induction of maintenance of Th1 type of antiviral immune response could benefit from such an approach. In addition, cross-linking of the chemokine receptors could also facilitate limited entry of HIV-1 into such cells because of downmodulation of the HIV-1 receptors. Such an approach has already been initiated, and preliminary studies have shown that inactivated HIV-1–pulsed DCs generated from CCR5– and CXCR4–cross-linked monocytes could trigger highly potent human anti–HIV-1 T-cell responses using our hu-PBL-SCID mouse model. These studies are currently in progress.


   Acknowledgments
 
We thank the NIH AIDS Research and Reference Reagent Program for supplying IL-2.


   Footnotes
 
This work was supported by grants from a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; Research on HIV/AIDS and Health Sciences focusing on Drug Innovation from the Ministry of Health, Labor and Welfare of Japan; and the Japan Human Science Foundation.

Received for publication November 13, 2005. Accepted for publication January 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. van Furth R, Cohn ZA. The origin and kinetics of mononuclear phagocytes. J Exp Med 128:415–435, 1968.[Abstract]
  2. Warren MK, Vogel SN. Bone marrow-derived macrophages: development and regulation of differentiation markers by colony-stimulating factor and interferons. J Immunol 134:982–989, 1985.[Abstract]
  3. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9:271–296, 1991.[Medline]
  4. Zhou LJ, Tedder TF. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc Natl Acad Sci U S A 93:2588–2592, 1996.[Abstract/Free Full Text]
  5. Imhof BA, Aurrand-Lions M. Adhesion mechanisms regulating the migration of monocytes. Nat Rev Immunol 4:432–444, 2004.[Medline]
  6. Mellado M, Rodriguez-Frade JM, Manes S, Martinez AC. Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation. Annu Rev Immunol 19:397–421, 2001.[Medline]
  7. Pulendran B, Palucka K, Banchereau J. Sensing pathogens and tuning immune responses. Science 293:253–256, 2001.[Abstract/Free Full Text]
  8. Donaghy H, Pozniak A, Gazzard B, Qazi N, Gilmour J, Gotch F, Patterson S. Loss of blood CD11c(+) myeloid and CD11c(–) plasmacytoid dendritic cells in patients with HIV-1 infection correlates with HIV-1 RNA virus load. Blood 98:2574–2576, 2001.[Abstract/Free Full Text]
  9. Soumelis V, Scott I, Gheyas F, Bouhour D, Cozon G, Cotte L, Huang L, Levy JA, Liu YJ. Depletion of circulating natural type 1 interferon-producing cells in HIV-infected AIDS patients. Blood 98:906–912, 2001.[Abstract/Free Full Text]
  10. Urban BC, Ferguson DJ, Pain A, Willcox N, Plebanski M, Austyn JM, Roberts DJ. Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400:73–77, 1999.[Medline]
  11. Fugier-Vivier I, Servet-Delprat C, Rivailler P, Rissoan MC, Liu YJ, Rabourdin-Combe C. Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells. J Exp Med 186:813–823, 1997.[Abstract/Free Full Text]
  12. Fong L, Engleman EG. Dendritic cells in cancer immunotherapy. Annu Rev Immunol 18:245–273, 2000.[Medline]
  13. Lu W, Arraes LC, Ferreira WT, Andrieu JM. Therapeutic dendritic-cell vaccine for chronic HIV-1 infection. Nat Med 10:1359–1365, 2004.[Medline]
  14. Garcia F, Lejeune M, Climent N, Gil C, Alcami J, Morente V, Alos L, Ruiz A, Setoain J, Fumero E, Castro P, Lopez A, Cruceta A, Piera C, Florence E, Pereira A, Libois A, Gonzalez N, Guila M, Caballero M, Lomena F, Joseph J, Miro JM, Pumarola T, Plana M, Gatell JM, Gallart T. Therapeutic immunization with dendritic cells loaded with heat-inactivated autologous HIV-1 in patients with chronic HIV-1 infection. J Infect Dis 191:1680–1685, 2005.[Medline]
  15. Lu W, Wu X, Lu Y, Guo W, Andrieu JM. Therapeutic dendritic-cell vaccine for simian AIDS. Nat Med 9:27–32, 2003.[Medline]
  16. Yosida A, Tanaka R, Murakami T, Takahashi Y, Koyanagi Y, Nakamura M, Ito M, Yamamoto N, Tanaka Y. Induction of protective immune responses against R5 human immunodeficiency virus type 1 (HIV-1) infection in hu-PBL-SCID mice by intrasplenic immunization with HIV-1–pulsed dendritic cells: possible involvement of a novel factor of human CD4+ T-cell origin. J Virol 77:8719–8728, 2003.[Abstract/Free Full Text]
  17. Lapenta C, Santini SM, Logozzi M, Spada M, Andreotti M, Di Pucchio T, Parlato S, Belardelli F. Potent immune response against HIV-1 and protection from virus challenge in hu-PBL-SCID mice immunized with inactivated virus-pulsed dendritic cells generated in the presence of IFN-alpha. J Exp Med 198:361–367, 2003.[Abstract/Free Full Text]
  18. Wu L, Dakic A. Development of dendritic cell system. Cell Mol Immunol 1:112–118, 2004.[Medline]
  19. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 392:245–252, 1998.[Medline]
  20. D’Amico A, Wu L. The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3. J Exp Med 198:293–303, 2003.[Abstract/Free Full Text]
  21. Romani N, Reider D, Heuer M, Ebner S, Kampgen E, Eibl B, Niederwieser D, Schuler G. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods 196:137–151, 1996.[Medline]
  22. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179:1109–1118, 1994.[Abstract/Free Full Text]
  23. Tuyaerts S, Noppe SM, Corthals J, Breckpot K, Heirman C, De Greef C, Van Riet I, Thielemans K. Generation of large numbers of dendritic cells in a closed system using Cell Factories. J Immunol Methods 264: 135–151, 2002.[Medline]
  24. Elkord E, Williams PE, Kynaston H, Rowbottom AW. Human monocyte isolation methods influence cytokine production from in vitro generated dendritic cells. Immunology 114:204–212, 2005.[Medline]
  25. Tanaka R, Yoshida A, Murakami T, Baba E, Lichtenfeld J, Omori T, Kimura T, Tsurutani N, Fujii N, Wang ZX, Peiper SC, Yamamoto N, Tanaka Y. Unique monoclonal antibody recognizing the third extracellular loop of CXCR4 induces lymphocyte agglutination and enhances human immunodeficiency virus type 1-mediated syncytium formation and productive infection. J Virol 75:11534–11543, 2001.[Abstract/Free Full Text]
  26. Grage-Griebenow E, Flad HD, Ernst M. Heterogeneity of human peripheral blood monocyte subsets. J Leukoc Biol 69:11–20, 2001.[Abstract/Free Full Text]
  27. Ohteki T, Fukao T, Suzue K, Maki C, Ito M, Nakamura M, Koyasu S. Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells. J Exp Med 189:1981–1986, 1999.[Abstract/Free Full Text]
  28. Tanaka Y, Zeng L, Shiraki H, Shida H, Tozawa H. Identification of a neutralization epitope on the envelope gp46 antigen of human T cell leukemia virus type I and induction of neutralizing antibody by peptide immunization. J Immunol 147:354–360, 1991.[Abstract]
  29. Inudoh M, Kato N, Tanaka Y. New monoclonal antibodies against a recombinant second envelope protein of hepatitis C virus. Microbiol Immunol 42:875–877, 1998.[Medline]
  30. Tozawa H, Andoh S, Takayama Y, Tanaka Y, Lee B, Nakamura H, Hayami M, Hinuma Y. Species-dependent antigenicity of the 34-kDa glycoprotein found on the membrane of various primate lymphocytes transformed by human T-cell leukemia virus type-I (HTLV-I) and simian T-cell leukemia virus (STLV-I). Int J Cancer 41:231–238, 1988.[Medline]
  31. Magaud JP, Sargent I, Mason DY. Detection of human white cell proliferative responses by immunoenzymatic measurement of bromo-deoxyuridine uptake. J Immunol Methods 106:95–100, 1988.[Medline]
  32. Patarroyo M, Prieto J, Beatty PG, Clark EA, Gahmberg CG. Adhesion-mediating molecules of human monocytes. Cell Immunol 113:278–289, 1988.[Medline]
  33. Davis GE. The Mac-1 and p150,95 beta 2 integrins bind denatured proteins to mediate leukocyte cell-substrate adhesion. Exp Cell Res 200:242–252, 1992.[Medline]
  34. MacIntyre EA, Roberts PJ, Jones M, Van der Schoot CE, Favalaro EJ, Tidman N, Linch DC. Activation of human monocytes occurs on cross-linking monocytic antigens to an Fc receptor. J Immunol 142:2377–2383, 1989.[Abstract]
  35. Lee C, Liu QH, Tomkowicz B, Yi Y, Freedman BD, Collman RG. Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signaling pathways. J Leukoc Biol 74:676–682, 2003.[Abstract/Free Full Text]
  36. Mellado M, Rodriguez-Frade JM, Manes S, Martinez AC. Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation. Annu Rev Immunol 19:397–421, 2001.[Medline]
  37. Vicente-Manzanares M, Rey M, Jones DR, Sancho D, Mellado M, Rodriguez-Frade JM, del Pozo MA, Yanez-Mo M, de Ana AM, Martinez AC, Merida I, Sanchez-Madrid F. Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis. J Immunol 163:4001–4012, 1999.[Abstract/Free Full Text]
  38. Dorsch M, Qiu Y, Soler D, Frank N, Duong T, Goodearl A, O’Neil S, Lora J, Fraser CC. PK1/EG-VEGF induces monocyte differentiation and activation. J Leukoc Biol 78:426–434, 2005.[Abstract/Free Full Text]
  39. Lauener RP, Goyert SM, Geha RS, Vercelli D. Interleukin 4 down-regulates the expression of CD14 in normal human monocytes. Eur J Immunol 20:2375–2381, 1990.[Medline]
  40. Bazil V, Strominger JL. Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes. J Immunol 147:1567–1574, 1991.[Abstract]
  41. Signoret N, Oldridge J, Pelchen-Matthews A, Klasse PJ, Tran T, Brass LF, Rosenkilde MM, Schwartz TW, Holmes W, Dallas W, Luther MA, Wells TN, Hoxie JA, Marsh M. Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4. J Cell Biol 139:651–664, 1997.[Abstract/Free Full Text]
  42. Signoret N, Rosenkilde MM, Klasse PJ, Schwartz TW, Malim MH, Hoxie JA, Marsh M. Differential regulation of CXCR4 and CCR5 endocytosis. J Cell Sci 111:2819–2830, 1998.[Abstract]
  43. Xiao X, Wu L, Stantchev TS, Feng YR, Ugolini S, Chen H, Shen Z, Riley JL, Broder CC, Sattentau QJ, Dimitrov DS. Constitutive cell surface association between CD4 and CCR5. Proc Natl Acad Sci U S A 96:7496–7501, 1999.[Abstract/Free Full Text]
  44. Rodriguez-Frade JM, del Real G, Serrano A, Hernanz-Falcon P, Soriano SF, Vila-Coro AJ, de Ana AM, Lucas P, Prieto I, Martinez-A C, Mellado M. Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor. EMBO J 23:66–76, 2004.[Medline]
  45. Ugolini S, Moulard M, Mondor I, Barois N, Demandolx D, Hoxie J, Brelot A, Alizon M, Davoust J, Sattentau QJ. HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4. J Immunol 159:3000–3008, 1997.[Abstract]
  46. Cosentino G, Soprana E, Thienes CP, Siccardi AG, Viale G, Vercelli D. IL-13 down-regulates CD14 expression and TNF-alpha secretion in normal human monocytes. J Immunol 155:3145–3151, 1995.[Abstract]
  47. Juliano RL, Haskill S. Signal transduction from the extracellular matrix. J Cell Biol 120:577–585, 1993.[Free Full Text]
  48. Jacob SS, Shastry P, Sudhakaran PR. Monocyte-macrophage differentiation in vitro: modulation by extracellular matrix protein substratum. Mol Cell Biochem 233:9–17, 2002.[Medline]
  49. Hamilton JA. CSF-1 signal transduction. J Leukoc Biol 62:145–155, 1997.[Abstract]
  50. Varticovski L, Druker B, Morrison D, Cantley L, Roberts T. The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase. Nature 342:699–702, 1989.[Medline]
  51. Carbonneil C, Aouba A, Burgard M, Cardinaud S, Rouzioux C, Langlade-Demoyen P, Weiss L. Dendritic cells generated in the presence of granulocyte-macrophage colony-stimulating factor and IFN-alpha are potent inducers of HIV-specific CD8 T cells. AIDS 17: 1731–1740, 2003.[Medline]
  52. Mohty M, Vialle-Castellano A, Nunes JA, Isnardon D, Olive D, Gaugler B. IFN-alpha skews monocyte differentiation into Toll-like receptor 7-expressing dendritic cells with potent functional activities. J Immunol 171:3385–3393, 2003.[Abstract/Free Full Text]
  53. Nagai T, Devergne O, Mueller TF, Perkins DL, van Seventer JM, van Seventer GA. Timing of IFN-beta exposure during human dendritic cell maturation and naive Th cell stimulation has contrasting effects on Th1 subset generation: a role for IFN-beta-mediated regulation of IL-12 family cytokines and IL-18 in naive Th cell differentiation. J Immunol 171:5233–5243, 2003.[Abstract/Free Full Text]
  54. Dauer M, Obermaier B, Herten J, Haerle C, Pohl K, Rothenfusser S, Schnurr M, Endres S, Eigler A. Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors. J Immunol 170:4069–4076, 2003.[Abstract/Free Full Text]
  55. Brossart P, Grunebach F, Stuhler G, Reichardt VL, Mohle R, Kanz L, Brugger W. Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte-macrophage colony-stimulating factor. Blood 92:4238–4247, 1998.[Abstract/Free Full Text]
  56. Buelens C, Bartholome EJ, Amraoui Z, Boutriaux M, Salmon I, Thielemans K, Willems F, Goldman M. Interleukin-3 and interferon beta cooperate to induce differentiation of monocytes into dendritic cells with potent helper T-cell stimulatory properties. Blood 99:993–998, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Exp. Biol. Med.Home page
L. F. Zhang, K. Okuma, R. Tanaka, A. Kodama, K. Kondo, A. A. Ansari, and Y. Tanaka
Generation of Mature Dendritic Cells with Unique Phenotype and Function by In Vitro Short-Term Culture of Human Monocytes in the Presence of Interleukin-4 and Interferon-{beta}
Experimental Biology and Medicine, June 1, 2008; 233(6): 721 - 731.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited