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Department of Molecular Biology, Molecular Immunology Unit, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-treated,
Ag-loaded macrophages induced a primary T cell response, indicated by
the occurrence of a proliferative response in vitro after restimulation
of splenocytes with Ag. Analysis of the accompanying cytokine secretion
revealed high numbers of IFN--producing Th1 cells and only a few
IL-4-secreting Th2 cells. This dominance of Th1 cells had functional
implications, reflected in the high titer of Th1 cell-dependent IgG2
Abs and the absence of IgG1, characteristic of humoral immunity.
Moreover, administration of Ag-loaded macrophages to mice with an
ongoing Th1/Th2 response resulted in a complete suppression of IgG1
production, whereas IgG2 levels remained unaffected. These results
demonstrate that macrophages exert APC activity in the organism,
strongly skew primary responses to cellular immunity, and in addition
suppress an already generated Th2-dependent humoral response, thus
characterizing these cells as Th1-oriented APC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
TNF-ß, contribute to cellular immunity, whereas Th2 lymphocytes,
mainly involved in humoral immunity, produce IL-4, IL-5, and IL-10.
Numerous examples of the consequences on disease outcome of skewed Th1
to Th2 ratios have been reported. Polarized Th2 responses have been
implicated in pathologic situations, such as after
Leishmania major (2, 3), human leprosy (4), or
mycotic infections (5). The contribution of Th1 cells relative to Th2
cells to a developing autoimmune response determines to a large extent
whether or not this response will lead to a clinical disease (6, 7, 8).
Also the chronic autoimmune graft-vs-host disease can be prevented by
switching a Th2 to a Th1 response by administration of IFN- at the
time of cellular transfer (9). Moreover, the presence of activated
tumor-infiltrating lymphocytes, characterized by a predominant type 2
cytokine production, has been linked to the inefficiency of the immune
response to a human glioma (10). This lack of efficiency of type 2
cytokines can be attributed to the fact that they do not promote a
tumoricidal immune response and therefore do not counteract the growth
of the tumor.
Clearly, parameters that control Th1/Th2 development may play a crucial
role in the susceptibility or resistance to a particular disease,
especially because individual Th0 cells appear to be capable of
differentiating into either T cell subset. Besides parameters such as
MHC haplotype (11), dose and nature of the Ag (12, 13), and the route
of Ag administration (14), the availability of IFN- and IL-12 as
opposed to IL-4 is decisive for the maturation to Th1 or Th2, both in
vitro and in vivo (15). Initial sources of IL-12 and IFN- are mainly
cells that are part of the innate immune system, namely macrophages,
dendritic cells, and NK cells. The cells responsible for the initial
production of IL-4 are less well defined and apparently include the
naive T cells themselves, induced by IL-6 (16). Because it is likely
that most in vivo responses do not take place in a milieu with
sufficient levels of cytokines, professional APCs may steer Th0
maturation to Th1 or Th2 by providing, besides the ligands for the TCR
and costimulatory receptors, also the necessary cytokines. Accordingly,
dendritic cells seem to induce preferentially the development of Th1
cells (17). However, it has also been described that dendritic cells
regulate both cellular and humoral immune responses (18). B cells, on
the other hand, seem to support the induction and expansion of Th2
cells (19). Finally, the involvement of macrophages in initiating
cognate immunity remains elusive. Although macrophages are dedicated
APCs in vitro, they exert this activity only after treatment with
IFN- and appear to be mainly involved in inflammation. However,
macrophages are an important source of IL-12 and might favor the
development of Th1 cells (20). This is supported by the observation
that macrophage depletion in mice shifts an expected Th1 response to a
Th2 response (21). This leaves open the question of whether the
involvement of macrophages is limited to that of a source of
environmental IL-12 or, on the contrary, also includes presentation of
Ag in the role of dedicated APC. In this report, we approached this
question by injecting clonal macrophages, appropriately loaded in vitro
with soluble protein, into syngeneic mice and by analyzing the specific
T cell response raised in vivo. Taken together, our data indicate that
macrophages efficiently elicit cellular immunity, selectively suppress
an already generated Th2-dependent humoral response, and hence behave
in the organism as a Th1-oriented, dedicated APC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from the Broekman Instituut (Eindhoven, The Netherlands). All mice were 9 to 14 wk old at the time of the experiments.
Immortalization of macrophages
Macrophages were immortalized as described (22). Briefly, primary cultures from spleens were plated at a density of 106 cells/ml. The cells were grown in RPMI 1640 (Life Technologies, Paisley, U.K.), supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (1 mM), and 2-ME (5 x 10-5 M). One day after seeding, cells were infected with VN11 retrovirus released by N11 producer cells (provided by Dr. P. Ricciardi-Castagnoli, CNR Center of Cytopharmacology, Milan, Italy). Briefly, 0.5 ml of N11 fresh supernatant from a 24-h subconfluent culture was filtered on 0.22-µm-pore size filters (Costar, Cambridge, MA), diluted 1/1 with complete medium containing 10 µg/ml Polybrene (Sigma Chemical, St. Louis, MO), and added to the primary cultures for 1 h at 37°C. Once established, the cells were cloned by limiting dilution.
Immunofluorescence
The phenotype of the cell clones was determined by indirect
immunofluorescence on live cells using a set of mAbs. Primary Abs were
R-phycoerythrin-conjugated anti-Mac-1 (CD11b), anti-CD14,
anti-CD18, anti-FcRII (CD32), anti-CD71 (PharMingen, San
Diego, CA), anti-Mac-2 (Cedarlane Laboratories, Hornby, Canada),
anti-BM-8, anti-ER-MP58 (BMA Biochemicals, Augst, Switzerland),
anti-F4/80, anti-CD40, biotinylated
anti-I-Ab (Serotec, Oxford, U.K.), anti-B7-1
(CD80) and anti-B7-2 (CD86) (a gift of Dr. K. Thielemans, Medical
School, Free University of Brussels, Brussels, Belgium).
FITC-conjugated goat anti-rat IgG (Life Technologies) was used as a
secondary Ab, except for detection of anti-B7-1 and biotinylated
Abs, where FITC-conjugated goat anti-hamster (Sera-Lab, Crawley
Down, U.K.) and streptavidin (Serotec), respectively, were used. Where
mentioned, cells were pretreated for 16 h with 400 U/ml of murine
rIFN- or with 10 µg/ml LPS. Analysis was performed using a
FACSCalibur cytofluorometer (Becton Dickinson, Sunnyvale, CA).
Phagocytosis of erythrocytes
SRBC were opsonized by mixing 1 volume of fresh SRBC (10% suspension; Sigma) with an equal volume of appropriately diluted anti-SRBC antiserum (Sigma) for 30 min at room temperature. Then the erythrocytes were washed, plated on monolayers of macrophages at a ratio of 50:1, and incubated at 37°C for 1 h in RPMI 1640 supplemented with 10% FBS. After removal of free SRBC by medium exchange and lysis by osmotic shock, the cells were fixed and stained with May-Grünwald (E. Merck, Darmstadt, Germany) and Giemsa (BDH, Poole, U.K.), after which they were microscopically examined for ingestion of SRBC.
Phagocytosis of fluorescent microspheres
Fluorescent microspheres were purchased from Duke Scientific (Palo Alto, CA) and opsonized with goat anti-mouse Ig Abs. Serial dilutions of the microspheres were made in PBS, sonicated for 30 s, and added to 105 cells. After incubation for 1 h at 37°C, unbound microspheres were separated from cells by passage through a Percoll gradient. Cells containing ingested microspheres were detected and quantified by flow cytometry. To distinguish membrane binding from internalization, a negative control was included consisting of samples incubated at 4°C. This temperature prohibits active processes such as phagocytosis, but not membrane binding.
Cytokine determination
Macrophages were harvested from adherent cultures grown in
tissue culture flasks using an enzyme-free cell dissociation buffer
(Life Technologies) and plated at a cell density of 3 x
104/200 µl in flat-bottom 96-microwell Falcon plates
(Becton Dickinson). After 24 h, cells were stimulated with 20
µg/ml LPS or 200 U/ml IFN- for 2 more days, and the culture fluid
was collected.
IL-1 and IL-6 levels were quantified by measuring the proliferation of cell lines D10(N4)M (23) and 7TD1 (24), respectively. TNF production was determined using the WEHI 164 clone 13 assay (25). IL-12 levels were determined by sandwich ELISA (BioSource International, Camarillo, CA), which detects both the heterodimeric IL-12 protein and the free p40 subunit.
T-HA proliferation assay
The CD4+ T cell clone T-HA, which is specific
for hemagglutinin (HA)3 and
is restricted to I-Ab, was developed in our laboratory by
immunization of C57BL/6 mice with 10 µg of HA and 0.1 ml of Ribi
adjuvant (Ribi Immunochem Research, Hamilton, MT), followed by
immunization with 3 µg of HA 3 weeks later. Five days after this
boost immunization, lymph nodes were isolated, and 3 x
107 cells were stimulated in vitro with 0.5 µg/ml HA in
25-cm2 culture flasks. On day 4, 10 U/ml murine IL-2 from
PMA-stimulated EL4.IL-2 cells were added to the cultures. After two
additional biweekly restimulations with 0.5 µg/ml HA and APC, a pool
of optimally HA-reactive T lymphocytes was obtained. T-HA cells were
maintained long term in vitro by biweekly restimulation in
25-cm2 culture flasks with 200 ng/ml HA and 7 x
107 syngeneic spleen cells from C57BL/6 mice (3000 rad
-irradiated). On day 2, 30 IU/ml of human rIL-2 were added, after
which T cells were further cultured and expanded by medium renewal and
IL-2 addition every 4 days. The cytokine secretion profile of
Ag-stimulated T-HA cells was typical of Th1 cells, namely, production
of IL-2 and IFN-, and lack of IL-4.
The Ag HA is the major surface glycoprotein of influenza virus and was prepared by digestion of purified X47 virus (A/Victoria/3/75 (H3N2)) with bromelain (26), purified by ion exchange on a DEAE column (Pharmacia Biotech, Uppsala, Sweden), and revealed as a single band on a silver-stained SDS-PAGE. The HA preparations obtained were endotoxin free.
Mf4/4 macrophages were seeded in 96-well flat-bottom microtiter plates
at a density of 2 x 104 cells/well. After 24 h,
the indicated concentrations of HA were added in combination with
IFN- (400 U/ml) or LPS (10 µg/ml), after which the cells were
further cultured overnight. The following day, macrophages were treated
with 50 µg/ml mitomycin C (Duchefa Biochemie, Haarlem, The
Netherlands) for 90 min at 37°C and thoroughly washed; 1 x
104 T-HA T cells were added to each well together with 40
µM indomethacin (Sigma) and 1248 U/ml catalase (Sigma). After 72
h, 0.5 µCi/well of [3H]TdR (Amersham Life Science,
Amersham, U.K.) was added for an additional 16-h culture. Cells were
harvested on glass fiber filters, and the incorporated radioactivity
was measured by liquid scintillation in a TopCount (Packard Instrument
Co., Meriden, CT). Results are means of triplicate cultures.
Immunization and proliferative response of immunized spleen cells
Mice were immunized against HA by i.p. injection of 2.5 µg HA,
dissolved in 200 µl of PBS or emulsified in 0.1 ml of Ribi adjuvant.
Alternatively, mice were injected with Mf4/4 macrophages presenting
HA-derived peptides. The latter were derived from Mf4/4 cultures
maintained for 48 h in FBS-free medium supplemented with 10 mg/l
insulin, 5.5 mg/l transferrin and 6.7 µg/l sodium selenite (ITS; Life
Technologies), to which 400 U/ml IFN- and 1 µg/ml HA were added
for the last 24 h. The treated cells were harvested, washed
extensively with PBS to remove free proteins, and immediately injected.
Repeated injections were given at biweekly intervals. Mice immunized
with Ribi adjuvant received an emulsion of HA and 25 µg
monophosphoryl lipid A (Ribi Immunochem Research) as adjuvant in the
boost injection. Unless otherwise mentioned, experimental groups
consisted of two animals. The proliferative response to HA of the
immunized spleen cells was assayed in 96-well flat-bottom microtiter
plates as described above. Briefly, the immunized mice were killed,
their spleens were removed and the splenocytes were seeded at 2 x
105 cells/well. Unless otherwise mentioned, 1 µg/ml
HA was added to the cultures. After 72 h, cell proliferation was
measured by [3H]TdR incorporation. Immunization
experiments were repeated at least once.
Cytokine assay
The frequency of Ag-induced T cells producing IFN- or IL-4
was determined with the enzyme-linked immunospot (ELISPOT) technique
according to the supplier’s protocol (PharMingen). Groups of mice were
immunized i.p. with HA-loaded macrophages, 2.5 µg of HA in PBS, 2.5
µg of HA emulsified in 0.1 ml of Ribi adjuvant or PBS as placebo,
followed by a second injection after 2 wk. Fourteen days after
immunization, 5 x 107 splenocytes were
stimulated ex vivo with 1 µg/ml HA in 5 ml of complete medium for
24 h. Viable cells were recovered from the cultures by passage
through a Histopaque 1077 density gradient (Sigma-Aldrich Co., Irvine,
U.K.). They were washed, seeded in nitrocellulose bottomed 96-well
Millititer HA plates (Millipore, Bedford, MA) at a density of 4 x
105 cells/well, and cultured for an additional 24 h.
Anti-IFN- and anti-IL-4 capturing and biotinylated detection
mAbs were purchased from PharMingen. Spots were visualized using
avidin-peroxidase and 3-amino-9-ethylcarbazole (Sigma) and were
microscopically counted. The frequency of cytokine-secreting cells was
derived from the increment of number of spots detected with immunized
vs placebo-treated splenocytes. No spots were detected in unstimulated
cultures without HA.
Determination of anti-HA Ab titer and isotype by indirect ELISA
Blood samples were taken and sera prepared 14 days after the last immunization. The sera were serially diluted in Maxisorp 96-well plates (Nunc, Roskilde, Denmark) previously coated with HA by overnight incubation at 4°C with a 0.5 µg/ml stock solution of the Ag. Bound Ab was detected with goat anti-mouse isotype-specific Abs (anti-IgG, anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgM; Sigma) using alkaline phosphatase-conjugated rabbit anti-goat IgG as detecting Ab (Sigma). Serum samples were collected from two mice per group and analyzed individually.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether macrophages were able to prime T cells in
vivo, macrophages isolated from the spleen of C57BL/6 mice were
immortalized. This provides the advantage over freshly isolated cells
of an unlimited source of functionally and phenotypically homogeneous
cell populations. Immortalization was conducted as described previously
by infection of spleen cell suspensions with VN11 retrovirus (22). This
resulted in the establishment of various cell lines exhibiting
macrophage features. Among these, clone Mf4/4 is a good representative
and was used throughout our additional experiments. Phenotypic analysis
of Mf4/4 revealed the presence of BM-8, a macrophage marker, as well as
of F4/80, Mac-1 (CD11b) and Mac-2, which are expressed only by mature
macrophages (Fig. 1
). The latter
characteristic was confirmed by the absence of the immature macrophage
marker ER-MP58. In addition, the cells expressed high levels of
FcRII (CD32) and CD14, both absent on dendritic cells (27), the
transferrin receptor CD71, and the adhesion molecule CD18.
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R-mediated
phagocytosis of SRBCs (28) and opsonized fluorescent microspheres,
respectively (data not shown), as well as secretion of IL-1, IL-6,
IL-12, and TNF-
after treatment with LPS (Table I). These cytokines were not detectable
in the culture fluid of cells stimulated with IFN-.
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). Treatment with
IFN- readily induced the expression of I-A, whereas the levels of
the constitutively expressed markers B7-1, B7-2 and CD40 remained
nearly constant. Stimulation with LPS resulted in a weak induction of
I-A, but in a strong, 5-fold increase in CD40 expression. To verify
whether the induction of an APC+ phenotype
(I-A+, B7-1+, B7-2+,
CD40+) by LPS, but especially by IFN-, had functional
implications, the capacity of the macrophages to activate T-HA cells
was assessed. T-HA is a Th1 cell clone which is specific for HA and is
restricted to I-Ab. Figure 3
shows the proliferative response of T-HA cells triggered by
mitomycin-C-treated Mf4/4 macrophages, previously loaded with
increasing amounts of HA and pretreated or not with IFN- or LPS.
Clearly, the induction of an APC+ phenotype by IFN-, but
not by LPS, was accompanied by the acquisition of APC activity. The
absence of proliferation after replacement of HA with hen egg white
lysozyme demonstrated that the observed response was Ag specific (data
not shown).
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in regulating this feature by
increasing the expression level of I-Ab MHC molecules,
thereby converting the macrophage to a dedicated APC. Injection of Mf4/4 macrophages, ex vivo loaded with HA, generates a primary anti-HA T cell response
To assess whether mature macrophages, induced to exert APC
activity by in vitro pretreatment with IFN- in the presence of an
optimal concentration of HA (1 µg/ml), also exerted this activity in
the animal, 1.5 x 106 activated and loaded Mf4/4
cells were injected i.p. into C57BL/6 mice. To reduce the level of
irrelevant proteins and to obtain a maximal presentation of relevant,
HA-derived peptides, the FBS present in the culture medium was replaced
with a mixture of insulin, transferrin, and selenium during coculture
with Ag. Two weeks after injection, the mice were killed, and their
spleen cells were assayed for the occurrence of a secondary anti-HA
T cell response (Fig. 4A). Splenocytes from
mice injected with HA-loaded macrophages elicited a pronounced
anti-HA proliferative response. This response was significantly
stronger than the one observed with spleen cells from mice injected
with soluble, intact HA and was comparable with the response of mice
injected with HA emulsified in adjuvant. Unlike HA-loaded macrophages,
injection of control macrophages did not prime the spleen cells for a
secondary anti-HA proliferative response, demonstrating the
dependence of the priming on HA-derived peptides. However, this does
not exclude the possibility that the peptides were presented to the
host T cells by endogenous APC, having acquired the antigenic peptides
on their MHC molecules by passive exchange or following uptake of
debris from dead Mf4/4 cells. Accordingly, we determined in
MHC-mismatched mice whether the secondary response was restricted by
the H-2 haplotype of the injected macrophages, namely, the recipient.
As shown in Figure 4B, injection of HA-loaded Mf4/4 cells
(H-2b) into H-2d BALB/c mice did not induce a
secondary, H-2d-restricted proliferative response,
triggered by BALB/c spleen APC. Nevertheless, both strains developed
comparable secondary responses after priming with free HA, emulsified
in adjuvant. We conclude therefore that the absence of a secondary
response in BALB/c splenocytes immunized with Mf4/4 macrophages can be
attributed to the H-2 mismatch between the APC involved in the primary
(H-2b) and secondary (H-2d) responses, thus
excluding nonspecific Ag reprocessing by the host. Hence, adoptively
transferred macrophages, loaded ex vivo with Ag, present the derived
antigenic peptides to T cells of the recipient and hereby prime the
animal against the Ag.
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After activation by Ag, naive CD4+ T cells
differentiate into Th1 and/or Th2 cells. To identify the
differentiation pathway that is predominantly activated following
immunization with macrophages, we analyzed the number of cells that
released Th1 vs Th2 cytokines. To raise a sufficient number of
HA-reactive T cells, mice were injected twice with HA-loaded Mf4/4
cells, free HA, HA emulsified in adjuvant, or PBS. Two weeks after the
second injection, splenocytes were restimulated in vitro for 24 h
with HA, presented by spleen APC. Live cells were recovered by passage
through Histopaque; the frequency of HA-reactive Th1 and Th2 cells was
assessed on the basis of IFN-- and IL-4-secreting cells,
respectively, using ELISPOT assays. As shown in Table II, mice injected twice with Ag-loaded
macrophages developed high numbers of IFN--producing Th1 cells,
comparable with the numbers obtained after immunization with HA
emulsified in adjuvant and significantly higher than the numbers
observed after immunization with free Ag. However, a remarkable
difference was observed for the Th2 cytokine IL-4. Mice vaccinated with
HA-loaded macrophages generated few IL-4-specific spots as compared
with animals that received HA emulsified in adjuvant. This differential
response is clearly reflected in the ratio of IFN-- to
IL-4-producing cells, where mice challenged with HA-loaded macrophages
exhibited a 10-fold increase relative to mice immunized with either
free or adjuvant-emulsified HA. Hence, macrophages shift the T cell
response to differentiation into Th1 cells, whereas free Ag induces a
mixed Th1 and Th2 response, independently of the presence or absence of
adjuvant and of the overall strength of the T cell response.
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Th1 and Th2 lymphocytes differentially affect the shift from the T
cell-independent IgM isotype to IgG isotypes in activated B
lymphocytes, supporting a switch to IgG2a/b and IgG1, respectively (31, 32). Accordingly, analysis of the isotype profile of anti-HA Abs
provides information regarding the preferential activation of Th1 cells
after immunization with Ag-loaded macrophages. Therefore, mice were
immunized with free HA or HA-loaded macrophages; 2 wk later, blood
samples were taken and serum from individual mice was prepared and
assayed. In contrast to HA-immunized mice, neither anti-HA IgM
(data not shown) nor IgG Abs could be detected in sera from mice
immunized once or twice with HA-loaded macrophages (Fig. 5). However, a second injection with free
HA instead of HA-loaded Mf4/4 cells induced high IgG titers, comparable
with those raised by two consecutive injections of free Ag and 10-fold
stronger than the titers raised by a single HA injection (Fig. 5).
Apparently, the absence of circulating Ag in the case of immunization
with HA-loaded macrophages prevented Ab production, although the Th
cells generated promoted IgG production, provided the B cells were
challenged with circulating Ag.
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A).
These data reveal a remarkable difference in the distribution of
anti-HA IgG subtypes: mice primed with free HA, whether or not
emulsified in adjuvant, developed IgG1 as well as IgG2a and IgG2b Abs,
independently of differences in the relative strength of the response.
Opposed to this, mice primed with loaded macrophages developed IgG2a
and IgG2b isotypes, but no IgG1. This remarkable IgG isotype
composition indicates a nearly exclusive activation of Th1 cells, in
agreement with the results obtained in vitro by ELISPOT.
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B). This result indicates that we are not dealing
with a clone-specific phenomenon. Macrophages suppress Th2-dependent IgG1 production
As free HA raised Th1- as well as Th2-dependent IgG responses, we
analyzed the impact of a subsequent immunization with loaded
macrophages on the bias of the immune response to IgG2 or IgG1
isotypes, respectively. To this end, mice were injected with free HA,
followed 2 wk later by injection of either PBS as a control, free HA,
or HA-loaded macrophages. Finally, all mice were rechallenged with free
HA, and the IgG isotype profile of the generated anti-HA Abs was
analyzed by ELISA (Fig. 7). As expected,
mice that had received three injections with free HA, or where the
second injection had been replaced with a placebo (PBS), generated
comparable titers of IgG1, IgG2a, and IgG2b anti-HA Abs. However,
substitution of the second injection by HA-loaded macrophages
completely abolished the IgG1 response, while the levels of IgG2a and
IgG2b remained unaffected. This pronounced and selective inhibition in
vivo of a single isotype indicates that, provided Ag presentation
occurs by macrophages, the humoral branch of the immune response is
completely and irreversibly suppressed. Furthermore, this result
demonstrates the feasibility of redirecting a mixed immune response to
a predominant cellular response by administering Ag as peptides,
presented by macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results demonstrate that syngeneic macrophages that have been
loaded ex vivo with exogenous protein, induced a primary immune
response characterized by a predominant activation of Th1 reactivity.
The macrophages used in these experiments were derived from a clonal,
immortalized population that both functionally and phenotypically
expressed features characteristic of mature macrophages. Thus, these
Mf4/4 cells expressed the surface molecules BM-8, F4/80, Mac-1, Mac-2,
and CD14 that have been described for mature macrophages; they exerted
receptor-mediated phagocytosis and produced IL-1, IL-6, IL-12, and TNF
in response to LPS, but not to IFN-. Moreover, the Mf4/4 cells
expressed increased levels of MHC class II Ags after treatment with
IFN- and concomitantly acquired the capacity to present exogenous Ag
to CD4+ T cells. These results demonstrate that, despite
their transformed state, the Mf4/4 cells retained their
macrophage-specific constitutive and inducible functions.
Injection into syngeneic mice of Ag-(HA)-loaded Mf4/4 cells generated a
primary T cell response. This was inferred by the presence of a
secondary proliferative response when the splenocytes were restimulated
with HA and the absence of such a response in naive splenocytes. This
proliferative response was observed only when the injected macrophages
were first treated with IFN- and cocultured with HA and when the
spleen APC, presenting the Ag in the secondary response, possessed the
same MHC haplotype as the injected macrophages. The latter finding
indicates that the induced immunity was a consequence of Ag
presentation by the injected macrophages rather than by endogenous APC
that acquired antigenic peptides by exchange of MHC class II-bound
peptides or by capture of membrane-bound HA.
Analysis of the cytokine secretion pattern by ELISPOT revealed that the
ratio of IFN- to IL-4-producing, HA-reactive cells was about 10-fold
higher in mice immunized with Ag-loaded macrophages than in mice
immunized with soluble Ag or Ag emulsified in adjuvant. Clearly, this
shift to a Th1 cytokine profile had functional implications. Thus, the
anti-HA Ab response induced by a subsequent immunization with
soluble HA contained, besides the T cell-independent IgM Abs, a
remarkably high titer of T cell-dependent IgG Abs. The latter belonged
to the IgG2 isotype, while the IgG1 isotype, typical of humoral
immunity, was absent. This characteristic IgG isotype profile was in
strong contrast to the development of, besides IgG2a and IgG2b, IgG1
anti-HA Abs in mice immunized with soluble HA or HA emulsified in
adjuvant. Similar results were obtained with macrophage clones derived
from an independent immortalization experiment. We therefore conclude
that mature macrophages, which were induced by IFN- to present
exogenous Ags, are potent inducers of Th1 reactivity and cellular
immunity.
As already mentioned above, mice injected with Ag-loaded macrophages generated a strong IgG response after a boost with soluble Ag. This result was quite unexpected due to the lack of B cell reactivity during priming, apparent from the absence of anti-HA Abs. Whereas the latter observation can be explained by the inaccessibility of the Mf4/4-bound antigenic peptides to the B cell receptor, the strength of the secondary, in fact primary B cell response indicates that the level of available T cell help, rather than a previous encounter with Ag of the reactive B cells, is critical for IgG production. This conclusion is of relevance for those situations where vaccination is required, but injection of free Ag is not desirable due to, for example, toxicity of the protein.
The observed exclusive induction of Th1-derived cellular immunity by
IFN--treated macrophages defines these cells as "Th1 APC."
Several mechanisms have been proposed for the selective induction of
Th1 or Th2 responses by Ag. Conboy et al. (35) demonstrated that the
genetic background of APC may influence T cells. However, since the
macrophage cell line shares its genetic background with (undefined)
endogenous APCs, the occurrence of a polarized vs a mixed T cell
response can be attributed to characteristics inherent to cells
presenting Ag, rather than to the genetic background of the mouse
strain. Certain characteristics of Mf4/4 cells point to a number of
possibilities. First, a recent report by DeKruyff et al. (36) describes
a CD40-dependent pathway for the induction of IL-12 during responses to
T cell-dependent Ags. Since IL-12 favors the development of Th1
responses (20, 37) and Mf4/4 cells express high levels of CD40
molecules (Fig. 2), it seems quite possible that this pathway
contributes to the bias to Th1 development. Our unpublished observation
that IL-12 was not induced by IFN-, but was produced in cultures
containing Ag-loaded Mf4/4 macrophages and Ag-specific Th1 cells, is
consistent with this pathway. Also, the absence of IL-1, a cytokine
necessary for proliferation of Th2 cells (38), may further amplify the
inclination to Th1 development. Finally, a number of reports implicate
B7 isoforms in the differential development of either Th cell type (39, 40). However, such a mechanism seems less likely considering that both
B7-1 and B7-2 are expressed at significant levels by IFN--treated
Mf4/4 cells. Clearly, further elaboration of this experimental model is
necessary to elucidate the pathway(s) by which macrophages promote
selective differentiation of Th0 cells into Th1.
Of particular interest is the observation that administration of Ag-loaded macrophages in between injections of free Ag resulted in the complete suppression of Th2-dependent IgG1 production. A similar, selective down-regulation has been observed after treatment with Ag-coupled splenocytes (41). Here the absence of costimulatory signals and the supposedly lesser dependence of Th1 cells on costimulation was proposed as a mechanism. However, considering the high expression level of costimulatory ligands on Mf4/4 macrophages and the lack of evidence in support of costimulatory preferences for restimulation of Th1 or Th2 subsets, it is tempting to speculate that a macrophage-specific feature is responsible for the apparently selective restimulation of Th1 memory cells. Accordingly, it cannot be excluded that a thus far unidentified molecule, expressed on the macrophage membrane, selectively activates memory Th1 cells and/or induces anergy or apoptosis in memory Th2 cells. Alternatively, IL-12 derived from Ag-loaded macrophages may have switched Th2 cells to Th1. Because this type of switch requires a low Ag density (3) and because macrophages, compared with dendritic cells, present less antigenic peptides due to lower expression levels of class II molecules, they may be more prone to induce such a switch.
Although the results described above have been obtained with
immortalized cell lines, the full retention of mature
macrophage-specific features by the cells supports the assumption that,
provided they are appropriately induced by IFN-, mature macrophages
are able to exert APC activity in the organism. Likely sources of
IFN- are NK cells and/or Ag-activated Th1 cells (42). Hereby, the
induced macrophages may promote Th1-dependent cellular immune
responses. In addition, the observed suppression of Th2 cell reactivity
without affecting and even promoting the Th1 subset opens perspectives
for treatment of various infective diseases in their acute phase, such
as leishmaniasis and tuberculosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. Grooten, Department of Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
3 Abbreviations used in this paper: HA, hemagglutinin; ELISPOT, enzyme-linked immunospot.
Received for publication November 10, 1997. Accepted for publication February 2, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2, IL-4 and IL-10 by CD4+ lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc. Natl. Acad. Sci. USA 88:7011. prevents Th2 cell-mediated pathology after neonatal injection of semiallogenic spleen cells in mice. J. Immunol. 153:2361.[Abstract]
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