The adjuvant monophosphoryl lipid A increases the function of antigen-presenting cells
Geneviève De Becker,
Véronique Moulin,
Bernard Pajak,
Claudine Bruck1,
Myriam Francotte1,
Clotilde Thiriart1,
Jacques Urbain and
Muriel Moser
Département de Biologie Moléculaire, Université Libre de Bruxelles, Rue des Prof. Jeener et Brachet 12, 6041 Gosselies, Belgium
1 Smith-Kline Beecham Biologicals, Rue de l'Institut 89, 1330 Rixensart, Belgium
Correspondence to:
M. Moser
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Abstract
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The induction of immune responses in vivo is typically performed with antigens administered in external adjuvants, like alum, complete Freund's adjuvant, LPS and, more recently, monophosphoryl lipid A (MPL). However, the role of the adjuvant is still poorly defined. The aim of this study was to test whether the MPL affects the function of antigen-presenting cells (APC) in vitro and in vivo. Antigen-pulsed APC [including macrophages, B cells and dendritic cells (DC)] were incubated or not with MPL, and their ability to sensitize naive T cells was tested in vitro and in vivo. The data show that MPL enhances the ability of macrophages and B cells to sensitize naive T cells, and confers to them the capacity to induce the development of Th1 and Th2. Administration of MPL i.v. in mice results in the redistribution of fully mature DC in the T cell area of the spleen. These observations suggest that MPL may induce an antigen-specific primary immune response by provoking the migration and maturation of DC that are the physiological adjuvant of the immune system.
Keywords: adjuvant, in vivo animal models, primary response, Th1, Th2
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Introduction
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The induction of antigen-specific primary immune responses in vivo usually requires the use of an adjuvant (1). The role of adjuvants is to provide the `on' signal that synergizes with antigen for the initiation of an optimal immune response. Numerous adjuvants have been used in rodents, and have been shown to affect quantitatively and qualitatively the immune response that ensues. In particular, the presence of an adjuvant increases the level of antibodies and the duration of the immune response, modifies the Th1/Th2 balance, and induces an anamnestic response.
However, most adjuvants have adverse side effects and are therefore not accepted in humans for routine vaccines (1). Recently, a method (2) was found to attenuate the extreme toxicity of lipopolysaccharide (LPS) (3), which is a potent adjuvant in vivo (4). The product obtained, referred to as monophosphoryl lipid A (MPL) (5), has retained the immuno-stimulating activities of the parent LPS molecule, but has a greatly attenuated toxicity. MPL has been proven to have adjuvant activity in both cellular and humoral effector arms of immunity, and has been administered safely to humans in various clinical vaccine trials (6). In mice, MPL is a potent adjuvant for protein antigens and favors the development of Th1-type responses, as assessed by production of IL-2, IFN-
and antibodies of IgG2a isotype (2).
The mechanism by which adjuvants co-initiate an immune response is still poorly understood. The role of adjuvants could be to provide the signal of `danger' which, as proposed by Janeway and Matzinger (7,8), is necessary to turn on the immune system (see below). Infection by Gram-negative bacteria can be detected with high sensitivity by the presence of LPS in their outer membrane (3). In this context, it is not surprising that LPS has potent adjuvanticity. Based on our previous studies showing that LPS induces the functional maturation of dendritic cells (DC) and their migration to T cell zones where immune responses are likely to occur (9), we hypothesized that LPS and MPL may act at the level of antigen presentation.
The population of cells able to process and present antigens in the context of class II MHC molecules is heterogeneous, and comprises B cells, macrophages and DC. Among them, cells of the dendritic family have the unique property to sensitize naive T lymphocytes and play a major role in the induction of primary immune responses in vitro and in vivo (10). We tested whether MPL would affect the antigen-presentation function of APC populations, and show indeed that LPS and MPL improve the capacity of B cells and macrophages to prime T and B cells, and induce the maturation of splenic DC in situ.
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Methods
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Mice, antigens and adjuvants
Female BALB/c mice, 68 weeks old, were purchased from Iffa-Credo (Bruxelles, Belgium) and housed in our pathogen-free animal facility.
The antigens used in this study are keyhole limpet hemocyanin (KLH) from Calbiochem-Novachem (San Diego, CA) and
-globulin from human blood (HGG), fraction II (Fluka Chemie, Buchs, Switzerland).
The 3-desacylated MPL was manufactured as described by Ulrich et al. (2) from culture of Salmonella minnesota R595. Briefly, LPS is obtained in soluble form by solvent extraction from the dried cell mass and the LPS residue is exposed to acid hydrolysis. This treatment causes the loss of the glycosidic phosphate at the 1 position and the inner core residues attached via the 6 position, generating 4'-MPL. This product is further exposed to mild alkaline treatment, which removes the fatty acyl residue attached to the 3 position.
LPS from Escherichia coli (serotype 055:B5) was purchased from Difco (Detroit MI).
Culture medium
The culture medium used for the isolation and antigen pulsing of APC was RPMI 1640 (Seromed, Biochem, Berlin, Germany) supplemented with 1% mouse serum (for in vivo injection) or 5% FCS (for mixed lymphocyte reaction assay) and additives (penicillin, streptomycin, non-essential amino acids, sodium pyruvate, 2-mercaptoethanol and L-glutamine; Flow ICN Biomedicals, Amersham, UK). Lymph node cells were cultured in Click's medium (Irvine, Santa Ana, CA) supplemented with 0.5% heat-inactivated mouse serum and additives.
APC
Purification.
DC were purified from spleens according to a procedure described by Crowley et al. (11). Briefly, spleens were digested with collagenase (CLSIII; Worthington Biochemical, Freehold, NJ), and separated into low- and high-density fractions on a BSA gradient (Bovuminar Cohn fraction V powder; Armour Pharmaceutical, Tarrytown, NY). Low-density cells were cultured for 2 h and the non-adherent cells were removed by vigorous pipetting. The same procedure was repeated with a shorter incubation (1 h) without serum. After overnight culture, non-adherent cells contained at least 90% DC, as assessed by morphology and specific staining using anti-CD11c mAb (N418). Peritoneal macrophages were purified from untreated mice injected i.p. with 10 ml cold sucrose (0.34 M). The peritoneal cells were harvested, cultured for 4 h and non-adherent cells were removed by vigorous pipetting. After overnight culture, the adherent cells were collected using a rubber policeman, and contained at least 90% of macrophages as assessed by morphology and specific staining using Mac-1 mAb.
Resting B cells.
Spleen cells were depleted of DC, macrophages and activated B cells by a passage over a Sephadex G10-column (Pharmacia, Uppsala, Sweden). The eluted cells were then depleted of T cells by treatment with anti-Thy1.2 mAb and complement, and cultured overnight. More than 90% of recovered cells were B cells as shown by staining with anti-mouse
light chain mAb.
Antigen pulsing.
The various populations of APC were cultured overnight in medium supplemented with mouse serum, additives and antigen (50 µg/ml of KLH or 100 µg/ml of HGG), in the presence or not of adjuvant (50 µg/ml MPL or 25 µg/ml LPS).
Immunization protocols
Humoral response.
Mice received an i.v. injection of 3x105 HGG-pulsed APC, treated or not with MPL in vitro. Five days later, all mice received a boost of 5 µg of soluble antigen i.v. (to activate B cells which recognize the antigen in its native form). The mice were bled 20 days after the antigen boost.
Cellular response.
KLH-pulsed APC, treated or not with LPS or MPL, were administered at a dose of 3x105 cells in a volume of 50 µl into the fore and hind footpads of syngeneic mice. The draining lymph nodes (popliteal and brachial) were harvested 5 days later.
Determination of antigen-specific antibody levels
Serum levels of antigen-specific antibodies were determined by ELISA according to standard procedures using polyclonal goat anti-mouse Ig reagent (Boehringer Mannheim, Mannheim, Germany) or isotype-specific rat mAb (12). Antibody titers were calculated based on linear regression analysis of the optical densities. Results are expressed as titers determined using the midpoint of the titration curves relative to an internal standard run in each assay.
In vitro assays
Mixed lymphocyte reaction.
Splenic T cells from CBA mice were depleted of adherent cells by passage over a Sephadex G10 column (Pharmacia) and cultured with
-irradiated splenic DC or resting B cells, or peritoneal macrophages isolated from BALB/c mice.
KLH-specific T cell response
. Lymph node cells (4x105) were cultured in round-bottom 96-well plates, with or without KLH (10 µg/ml). The proliferation was measured as thymidine incorporation during the last 1216 h of the 3 day culture. Supernatants from cultures were assayed for IL-2 after 24 h, for IL-4 after 48 h, for IFN-
after 72 h and for IL-5 after 96 h of incubation.
Lymphokine assays
Culture supernatants were assayed for IL-2 content by a standard bioassay using an IL-2-sensitive, IL-4-insensitive subline of the CTLL cell line. IFN-
was quantitated by a two-site ELISA using mAb F1 and Db-1, kindly provided by Drs Billiau and Herremans (KUL, Leuven, Belgium) and P. H. Van Der Meide (TNO Health Research, Rijswijk, The Netherlands) respectively. IL-4 and IL-5 were quantitated by two-site ELISA from Genzyme (Cambridge, MA) and PharMingen respectively.
Immunohistochemistry
Spleens were fixed for 3 days in Immunohistofix (B. Pajak et al., manuscript in preparation) followed by dehydration in a graded series of ethanol (30, 50, 70, 90 and 100%) for 30 min each at room temperature. Tissues were embedded in Immunohistowax (13), sectioned at 36 µm, de-embedded by washing in acetone for 5 min and transferred to PBS. The sections were treated for 30 min with Blocking Reagent (1% in PBS, from Boehringer) to saturate non-specific reactions sites. The endogenous peroxidase activity was neutralized by 3% H2O2 in PBS for 60 min, and the slides were stained with biotinylated anti-B7-2 and anti-CD4 mAb, followed by avidinbiotinphosphatase complex (Vectastain ABC kit; Vector, Burlingame, CA) and revealed with the alkaline phosphatase substrate kit III blue (Vector). Sections were further stained for CD11c expression. The excess of biotin from the first antibodies was blocked with the Vector blocking kit (Vector), and the sections were incubated with biotinylated anti-CD11c mAb, followed by avidinbiotinperoxidase complex and revealed with 3-amino-9-ethyl-carbazol tablets (Sigma, Bornem, Belgium).
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Results
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MPL up-regulates the immuno-stimulatory capacity of B cells and macrophages in vitro
We first tested the capacity of B cells, macrophages and DC to induce a primary response in vitro. The data in Fig. 1
(B) show that, in the absence of intentional stimulation, DC have the unique capacity to sensitize naive allospecific T lymphocytes in vitro. By contrast, T lymphocytes poorly proliferated when cultured with allogeneic B cells or macrophages in the same conditions. Treatment with MPL converted B cells (Fig. 1B
) and macrophages (Fig. 1A and B
) into potent APC, able to initiate a primary immune response in vitro, as assessed by enhanced proliferation of T cells stimulated by MPL-treated APC. The dose of 50 µg/ml of MPL was chosen for further studies, as it was the highest dose that strongly enhanced the immuno-stimulatory capacity of macrophages, without affecting their viability (not shown).

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Fig. 1. Treatment of DC, B cells and macrophages with MPL increases their capacity to sensitize naive T cells in vitro. (A) T cells (3x105) from CBA mice were cultured without (None) or with 2.5x104 peritoneal macrophages, treated (MPL) or not (none) with graded doses of MPL during overnight culture. Proliferation was measured as indicated in the text. The results represent the mean ± SD of triplicate cultures. (B) T cells (3x105) from CBA mice were cultured with various numbers of -irradiated DC, macrophages (Mac) or resting B cells (Br) from BALB/c mice. The APC populations were treated or not with MPL (50 µg/ml) in vitro. Proliferation was measured as indicated in the text. The proliferation of T cells alone (without APC) was 3767 ± 309 c.p.m. The results represent the mean ± SD of triplicate cultures. Similar data were obtained in four experiments performed.
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MPL-treated B cells and macrophages acquire the capacity to induce an antibody response, characterized by the production of IgG1 and IgG2a
To assess their function in vivo, unstimulated or MPL-treated cells were pulsed extracorporeally with HGG and 3x105 cells were injected i.v. into syngeneic recipients. Five days later, animals were injected with 10 µg antigen in saline (to activate B cells which recognize the antigen in its native form). The data in Fig. 2
show that in vivo administration of HGG-pulsed DC or macrophages induced a humoral response upon challenge with soluble antigen, as previously shown (14). By contrast, very low levels of specific antibodies were detected in the mice injected with HGG-pulsed B cells. Treatment with MPL strongly enhanced the capacity of B cells and macrophages to initiate an humoral response, and did not affect the humoral response induced by DC. We next analyzed the isotypic profile of the antibodies produced. Injection of antigen-pulsed DC induced the synthesis of specific IgG2a and IgG1 antibodies, whereas peritoneal macrophages triggered the production of IgG1 only, as previously shown (14). Treatment with MPL in vitro conferred to macrophages the capacity to induce high levels of IgG2a antibodies and to B lymphocytes the capacity to induce an antibody response characterized by the production of intermediate levels of IgG1 and IgG2a in vivo.

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Fig. 2. Isotype analysis of HGG-specific primary responses. Mice were injected i.v. with 3x105 DC, macrophages (Mac) or resting B cells (Br), pulsed (HGG) or not in vitro with 100 µg/ml of HGG. When indicated (MPL), APC were incubated overnight with MPL (50 µg/ml). All mice received a boost of 10 µg HGG i.v. 5 days later. Total specific response (Ig Tot) as well as specific antibodies of IgG1 and IgG2a isotypes were tested on day 20 as described in Methods. Serum were tested individually (three to four mice in each group). Data are shown as means ± SD (95% confidence). Data are representative of all experiments (three) performed, except the group injected with MPL-treated DC which represents the data of three mice in one experiment.
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MPL-treated B cells and macrophages have an increased capacity to prime antigen-specific T cells in vivo
The distinct isotypic profiles of the humoral responses suggested that untreated and MPL-treated DC, macrophages and B lymphocytes stimulated the differentiation of distinct Th subsets. We directly assessed the activation of Th cells by measuring the lymphokines produced by lymph node T cells from mice primed with antigen-pulsed APC populations. We have shown previously that priming of HGG-specific T cells required two injections of antigen-pulsed macrophages (15), whereas KLH-specific T cells were sensitized with one injection of APC (16). We therefore injected KLH-pulsed DC, macrophages or B cells, treated or not with MPL, into the footpads of syngeneic animals and harvested the draining lymph nodes 5 days later. The data in Fig. 3
(A) show that T cells were primed in all groups of mice, except in animals injected with unstimulated B lymphocytes, as assessed by proliferation upon re-stimulation in culture with KLH. Analysis of the lymphokines produced in the supernatants revealed that DC, incubated or not with MPL, induced the production of IFN-
, IL-4 and IL-5, although MPL-treated DC induced the secretion of higher levels of IFN-
and lower levels of IL-4 than untreated DC. Unstimulated macrophages triggered the production of IL-4 only, whereas MPL-treated macrophages induced the secretion of intermediate levels of IFN-
, high levels of IL-4 and IL-5. B lymphocytes poorly activated the proliferation and lymphokine secretion by lymph node cells, but, upon stimulation, acquired a superior capacity to induce antigen-specific proliferation and secretion of IFN-
, IL-5 and, to a lesser extent, IL-4. Collectively, these observations indicate that treatment of APC with MPL increases the immuno-stimulatory capacity of B cells and macrophages, and drives the development of cells that secrete IFN-
, IL-4 and IL-5.

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Fig. 3. Effect of MPL on the capacity of APC to sensitize T lymphocytes in vivo. DC, macrophages (Mac) and resting B cells (Br) were isolated from BALB/c mice, pulsed with KLH (50 µg/ml) in the presence or absence of MPL (50 µg/ml) or LPS (25 µg/ml), as indicated, and injected in the hind and fore footpads of syngeneic mice. Five days later, lymph nodes were harvested and cultured with KLH. Lymphokines in the supernatants and proliferation were measured as indicated in Methods. Data are shown as means of duplicate cultures and the error bars represent 1 SD from the mean. Similar results were obtained in three independent experiments
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Injection of MPL induces the migration and maturation of DC in vivo
We have shown previously that injection of microbial products, such as LPS, resulted in phenotypic and functional maturation of splenic DC and their redistribution in T cell areas (9). To test whether MPL would similarly affect the function of DC in vivo, we injected saline, 25 µg LPS or 50 µg MPL i.v., and monitored the phenotype and localization of splenic DC 6 h later by immunohistochemistry. The spleen sections were embedded in Immunohistowax and fixed according to a process recently described (13), and further stained for expression of CD11c and B7-2 molecules. The data in Fig. 4
show that the majority of N418+ cells (red staining) were detected in the marginal zone between red and white pulp, whereas a few cells were labeled in the T cell area (CD4 staining in blue) around the central arteriole (Fig. 4a
, ca). Injection of MPL (Fig. 4b and e
) or LPS (Fig. 4c and f
) induced the migration of most DC from the marginal zone to the area where T cells are located, around the central arteriole. Of note, the expression of the B7-2 molecule (blue staining in Fig. 4gl
) was strongly up-regulated on new migrants after MPL (Fig. 4h and k
) or LPS (Fig. 4i and l
) injection, as assessed by purple staining of DC stained for CD11c (in red) and B7-2 (in blue). By contrast, most DC in the spleen of mice injected with saline only do not express B7-2 (Fig. 4g and j
). Some DC located in the T cell area of MPL-treated mice remained CD11c+, B7-2, whereas the majority of DC in the spleen of LPS-injected mice expressed high levels of B7-2 (cf. Fig. 4k and l
).

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Fig. 4. MPL induces migration and maturation of splenic DC. Immunostaining of spleen sections of BALB/c mice treated 6 h previously with NaCl (left panels), 50 µg/ml MPL (middle panels) or 25 µg/ml LPS (right panels) i.v. (af) Sections were double stained with DC-specific mAb (anti-CD11c) in red and T-specific mAb (anti-CD4) in blue, the original magnification was x10 (ac) and x20 (df). (gl) Sections were double stained for CD11c (in red) and B7-2 (in blue), the original magnification was x10 (gi) and x20 (jl). Control includes section stained for B7-2 only (insert i). mz = marginal zone; ca = central arteriole. Three independent experiments were performed with similar results.
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Discussion
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The activation of antigen-specific CD4+ T cells requires at least two signals provided by the APC: the presentation of the antigen in a peptidic form in the groove of MHC class II molecules and the expression of co-stimulatory molecule(s). Importantly, there is evidence that most APC do not constitutively express co-stimulatory signals (17). Unstimulated B lymphocytes and macrophages poorly stimulate naive T cells in vitro and in vivo (1820). Purified DC appear to have high stimulatory capacity, but this property develops during a process of maturation that occurs spontaneously upon culture. Freshly isolated DC were indeed shown to express low levels of B7-1 and B7-2 (20). Furthermore, analysis of sections of various organs revealed that B7-1 and B7-2 staining was weak or negative in most non-lymphoid organs, and that high expression of B7-1 and B7-2 molecules was restricted to selected sites in lymphoid organs (21). Collectively, these data suggest that a crucial step for the induction of the immune response is the induction of co-stimulation.
The `danger' theory could provide some insights in the mechanism by which adjuvants initiate the immune response (8). Indeed, Liu and Janeway (16) showed that a variety of infectious agents, including bacteria and viruses, or their constituents can induce accessory cells to become co-stimulatory for CD4 T cells. In particular, LPS treatment rendered B cells and macrophages competent to induce clonal expansion of CD4 T cells in vitro. Matzinger further proposed that the immune system was able to discriminate danger (8). The sensor of the danger signal would be the APC that constitutes the first step in the adaptive immunity. In line with this hypothesis, we and others have shown that injection of LPS or Toxoplasma gondii extracts induced the maturation of DC that acquire the capacity to sensitize naive T cells (9,22). Similarly, the data presented herein indicate that treatment with MPL in vitro increases the immuno-stimulatory properties of DC, macrophages and B lymphocytes in vitro and in vivo. Unexpectedly, expression of B7-2 remained unchanged on B cells and macrophages upon MPL treatment in culture (data not shown). The increased immunogenicity of these cells could result from in vivo up-regulation of B7-2 on transferred cells (upon interaction with T cells?), or, alternatively, MPL-treated B cells or macrophages may express other co-stimulatory molecules (like heat stable antigen?).
Injection of MPL induces the migration of DC from the marginal zone of the spleen to the T cell area. New migrant DC expressed increased levels of MHC class II (not shown) and B7 co-stimulatory molecules. In addition, immunohistochemical analysis revealed an increase in B7-2 expression on cells located at the marginal zone following LPS or MPL injection (cf. Fig. 4h
, i with g). These cells are CD11c, Mac-1 (not shown) and could be marginal metallophils. B lymphocytes remained B7-2 in the same conditions. Collectively, these results suggest that injection of MPL primarily activates DC and possibly a subset of macrophages. Our in vitro data show that MPL could convert B cells and macrophages into potent APC, suggesting that higher dose(s) of MPL or a different kinetics would result in stimulation of all APC populations in vivo.
There is evidence that primary immune responses are induced in secondary lymphoid organs (23,24). The activation of naive T cells by cells that present the antigen and provide co-stimulatory signal(s) requires their physical interaction. As B cells, T cells, macrophages and DC are located in discrete sites in lymphoid organs, the induction of the immune responses is likely to imply a redistribution of cell populations. In particular, we have reported that LPS causes the migration and maturation of splenic DC from the marginal zone to the T cell areas (9), leading to co-localization of T lymphocytes and fully mature DC. The observations presented herein strongly suggest that MPL similarly provokes the redistribution of B7+ DC in the zone where T cells are located.
The population of Th cells is heterogeneous and includes at least two subsets that differ by the pattern of lymphokines they secrete (25). Th1 cells produce IL-2 and IFN-
, whereas Th2 lymphocytes produce IL-4, IL-5 and IL-10. Importantly, both subpopulations stimulate distinct effector functions: Th1 cells are mainly involved in the eradication of intracellular infectious pathogens, whereas Th2 lymphocytes are efficient to eliminate extracellular parasites. Our data show that MPL potentiates the functional properties of the APC subsets. Indeed, MPL-treated macrophages and B cells induce higher levels of IFN-
, IL-4 and IL-5, without altering the Th1/Th2 balance, and induce an humoral response that includes IgG1 and IgG2a antibodies. MPL-treated DC induce the development of cells producing higher levels of IFN-
, but lower levels of IL-4 and IL-5. The decreased production of Th2-type cytokines induced by MPL-treated DC could be due to direct inhibition of IL-4 secretion by elevated amounts of IFN-
, as reported (26), or, alternatively, MPL could inhibit the capacity of DC to induce the development of Th2 cells. Of interest, treatment of DC with MPL results in induction of an humoral response, characterized by high amounts of IgG2a and IgG1. The unchanged IgG1 production, despite lower levels of IL-4, is compatible with reports showing that secretion of antigen-specific antibodies of IgG1 isotype can be independent of IL-4 (27,28).
Our data show that MPL-treated DC promote a Th1-type response, whereas LPS-treated DC promote a Th0-type response. The differential effect of LPS and MPL on DC could be due to quantitative or qualitative differences. Additional experiments, using titrated amounts of LPS or MPL, would be required to address this issue. It is also possible that the core region or the o-specific chain could play a role in the stimulation of IL-4 production.
The mechanism by which LPS at the cell surface induces host cell activation across the cytoplasmic membrane is still poorly understood. Responses of myeloid cells to LPS require a plasma protein called LPS-binding protein (LBP) and the glycophosphatidylinositol-anchored membrane protein CD14, a major LPS receptor that lacks a transmembrane domain (2931). LPS monomers seem to be catalytically transferred by LBP to CD14 and a LPS transmembrane molecule would be responsible for initiation of cellular responses after interaction with LPSCD14 complex. Two putative co-receptors, Toll-like receptors (Tlr) 2 and 4, presumably transduce the LPS signal across the plasma membrane (3234). It has been shown recently that Tlr2 can be activated by lipid A alone (32), suggesting that Tlr2 may be involved in the activation of APC by MPL. In addition, experiments using LBP-deficient mice suggest that redundant or overlapping mechanisms may act to initiate cellular responses to LPS (29).
A new adjuvant system containing alum and MPL has been recently tested in adult volunteers, using as antigen hepatitis B surface antigen (6). The observations indicate that the formulation is safe and immunogenic. Importantly, seroconversion and seroprotection rates of 100% were reached earlier with SBAS4-HBV vaccine (containing recombinant hepatitis B surface antigen with a combination of aluminum salt and MPL) than with the commercially available recombinant hepatitis vaccine which contained the same recombinant antigen adsorbed on aluminum salt. In addition, higher in vitro proliferative responses were obtained in the same group, suggesting that MPL favors the sensitization of hepatitis B-specific T lymphocytes.
In conclusion, the observations presented herein suggest that the adjuvant MPL may act at the level of antigen presentation. Addition of MPL increases the immuno- stimulatory properties of B cells and macrophages in vitro, and induces the migration and functional maturation of DC in vivo. The redistribution of fully mature DC, that present antigens encountered in the periphery, in the T cell area of lymphoid organs is likely to constitute the very first step of the immune response.
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Acknowledgments
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We thank Dr Oberdan Leo for careful review of the manuscript; M. Brait, T. De Smedt and P. Mettens for valuable help; and G. Dewasme, M. Swaenepoel, F. Tielemans and P. Veirman for technical assistance. The Laboratory of Animal Physiology was supported by grants of the Fonds National de la Recherche Scientifique (FNRS)/Télévie, by the Fonds de la Recherche Fondamentale Collective, by the European Commission (CEC TMR Network contract FMRX-CT96-0053), and by the Belgian Programme on the Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. M. M. is Research Associate from the FNRS.
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Abbreviations
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APC antigen-presenting cells |
DC dendritic cells |
HGG -globulin from human blood |
KLH keyhole limpet hemocyanin |
LBP LPS-binding protein (LBP) |
LPS lipopolysaccharide |
MPL monophosphoryl lipid A |
Tlr Toll-like receptors |
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Notes
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The first two authors contributed equally to this work
Transmitting editor: J. Banchereau
Received 9 July 1999,
accepted 10 February 2000.
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