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Chain-Deficient Mice and Increased in FcRII-Deficient Mice1
Departments of
*
Genetics and Pathology and
Animal Development and Genetics, Uppsala University, Uppsala, Sweden; and
Department of Immunology, University Hospital Utrecht, Utrecht, The Netherlands
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RII, whereas the mechanism behind IgG-mediated
enhancement is unknown. We have investigated whether receptors for the
Fc part of IgG are required for responses to IgG/Ag. Mice lacking the
subunit of Fc receptors (FcRs) (FcR-/-), FcRII
(FcRII-/-), or FcRIII (FcRIII-/-)
were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP
complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As
expected, all subclasses enhanced the Ab-response to BSA in wild-type
mice. Enhancement was in the same order of magnitude in
FcRIII-/- mice (
177-fold of controls administered Ag
alone), whereas it was abrogated in FcR-/- mice and
augmented in FcRII-/- mice (5147-fold of controls).
The response to IgE/Ag complexes in FcR-/- and
FcRII-/- mice was similar to that seen for wild-type
mice, demonstrating that non-FcR-dependent responses were normal.
Our observations suggest that IgG/Ag complexes enhance Ab responses via
FcRs. Moreover, they reveal a strong negative regulation of Ab
responses to IgG/Ag exerted by FcRII. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs
and is most likely due to masking of epitopes by IgG Abs
(3). Whether suppression of immunological memory is also
Fc-independent or caused by negative signaling to the B cell via
IgG/Ag-induced co-cross-linking of surface Ig and the inhibitory
FcRIIB remains to be investigated. In contrast, complexes of IgG and
soluble protein Ags initiate much stronger in vivo Ab responses than Ag
alone (4, 5, 6, 7, 8), and the same monoclonal 2,4,6-trinitrophenyl
(TNP)-specific IgG can inhibit responses to SRBC-TNP while enhancing
responses to keyhole limpet hemocyanin (KLH)-TNP (5, 7).
In addition to IgG, IgE Abs are also able to up-regulate Ab responses
to soluble Ags. An absolute requirement for IgE-mediated enhancement is
a functional low-affinity receptor for IgE, FcRII (CD23)
(9, 10, 11). In vitro, IgE/Ag complexes are endocytosed by
CD23+ B cells, followed by efficient presentation
of Ag to T cells (12), and it is assumed that enhanced Ag
presentation explains the IgE-mediated up-regulation of in vivo Ab
responses. The mechanism behind IgG-mediated enhancement is less well
understood. IgG complexes fulfill their biological functions via
activating C or binding to FcRs. Involvement of the C system was
implicated in early studies, suggesting that IgG induced efficient
responses by increasing the localization of Ag in lymphoid follicles
(13). However, a non-C-activating mutant IgG was found to
be almost as efficient in enhancing the immune response as the
corresponding wild-type (wt) IgG (8). This finding
suggests another mechanism, possibly involving FcRs. There are three
classes of FcRs on murine leukocytes: FcRI (CD64), FcRII
(CD32), and FcRIII (CD16) (reviewed in Ref. 14).
FcRII and FcRIII are low-affinity receptors for IgG1, IgG2a, and
IgG2b. FcRI is a high-affinity receptor for monomeric IgG2a and was
recently reported to bind IgG3 also, although with moderate affinity
(15). In the present study, a panel of FcR-deficient
mice were immunized with IgG/Ag or IgE/Ag (as a non FcR-dependent
control), and the role of these receptors in IgG-mediated enhancement
of Ab responses was investigated. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The animals used herein were derived from
FcRIII-/- (16),
FcR-/- (17), and
FcRII-/- mice (18), all of the
H-2Ab haplotype. Because
H-2b mice have an
I-Ab-linked low responsiveness to IgE/BSA-TNP
(19, 20) and IgG/BSA-TNP (19), the mutant
mice were backcrossed to responder strains.
FcRIII-/- mice were crossed with CBA/J mice
(H-2Ak)(Charles River, Someren, The Netherlands)
or BALB/c mice (H-2Ad) (Charles River).
FcR-/- and
FcRII-/- mice were crossed with CBA/J mice
(H-2Ak) (Bommice, Bomholtgaard, Ry, Denmark). The
F1 generations were intercrossed, and homozygous
mutant H-2Ad and H-2Ak mice
as well as homozygous wt H-2Ak animals were
identified by PCR analysis of tail DNA. Offspring from these animals
were used in the experiments. Although the optimal strains would have
been fully congenic mice, the most important gene locus for the studied
Ab responses (I-A) was similar in mutant and wt animals. All mice were
bred and maintained at the Department of Genetics and Pathology and the
Department of Animal Development and Genetics (Uppsala University) or
at the Department of Immunology (University Hospital Utrecht).
Antigens
BSA, OVA, and TNP (picrylsulfonic acid/hydrate) were obtained from Sigma (St. Louis, MO). TNP was conjugated to BSA as described previously (20). BSA-TNP and OVA were stored in PBS at 4°C as sterile solution.
Antibodies
mAbs were derived from B cell hybridomas producing IgG1
anti-TNP (B8401H5(H5)), IgG2a anti-TNP (C4007B4(7B4)), IgG2b
anti-TNP (C1901B4(1B4) GKH-1-GORK(GORK)), and IgE anti-TNP
(IGELb4) and H5, 7B4, and 1B4 (6) as well as IGELb4
(21) have been described previously. GORK was a gift of
Dr. G. Köhler (Max Planck Institute, Freiburg, Germany). IgG was
purified on protein A- or protein G-Sepharose columns (Pharmacia,
Uppsala, Sweden), and IgE was purified on a Sepharose column coupled
with monoclonal rat anti-mouse 
. Abs were dialyzed against PBS,
sterile-filtered, and stored at -20°C. Protein concentrations were
determined by absorbance at 280 nm, assuming that an absorbance of 1.5
equals 1 mg/ml of Ab. The IgG subclass of the preparations was tested
by ELISA using subclass-specific antisera (data not shown).
Immunizations
Mice were immunized in the tail vein with 0.1 ml of a PBS solution containing BSA-TNP or BSA-TNP/Ab complexes formed by incubating BSA-TNP with TNP-specific Abs for 1 h at 37°C immediately before injection. OVA were included in the Ag mixtures as a specificity control.
Enzyme-linked immunosorbent assay
Blood was collected from the tail veins, and sera were tested using an IgG anti-BSA or IgG anti-OVA-specific ELISA (11). Statistical differences were determined by Student’s t test. p values are presented as: not significant, p > 0.05); *, p < 0.05; **, p < 0.01, or ***, p < 0.001. Stimulation indices (SI) were calculated as the geometrical mean of the experimental group divided by the geometrical mean of the control group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice
The subunit of Fc receptors (FcR) is associated
with FcRI, FcRIII, FcRI, and the TCR-CD3 complex. It is
important for receptor assembly and mediates activating signals via an
immunoreceptor tyrosine-based activation motif (ITAM) (reviewed in Ref.
14). Recently, the role of this receptor subunit has been
studied in mice lacking FcR (17). These animals do not
express FcRIII or FcRI, and their macrophages are unable to
phagocytose IgG2a/SRBC complexes, which also suggests a lack of
function of FcRI. FcR-/- mice have
defects in Ab-dependent cytotoxicity, hypersensitivity reactions, and
phagocytosis (17, 22, 23), whereas the Ab response after
immunization with KLH-4-hydroxy-3-nitrophenylacetyl in adjuvants is
normal (24).
To study the role of FcRs in IgG-mediated feedback enhancement,
FcR-/- and wt mice were immunized with
BSA-TNP alone or in complex with TNP-specific IgG1, IgG2a, or IgG2b
(Fig. 1
, A–C and
E–G). As expected, all IgG isotypes enhanced the Ab
response in wt mice. In FcR-/- mice,
enhancement by IgG1 and IgG2a was almost completely inhibited;
enhancement by IgG2b was reduced at early timepoints. The specificity
of Ab feedback enhancement was tested by including the
non-cross-reacting Ag OVA in all immunizations. IgG anti-OVA
responses above background levels were not detected in any of the
experiments described, and Abs injected alone were also unable to
induce a response (data not shown).
|
is also associated with the TCR-CD3 complex, we wanted
to exclude the possibility that the lack of Ab response was due to a
defect in T cell help leading to a general inability to produce Abs
after challenge with immune complexes. Therefore, mice were immunized
with IgE anti-TNP and BSA-TNP. Although IgE was reported to bind to
FcRII and FcRIII (25) as well as FcRI, these
receptors are unable to mediate responses to IgE/Ag complexes, as the
ability of IgE to up-regulate Ab responses is completely inhibited in
mice in which FcRII is blocked by mAbs (9, 11) and in
FcRII-/- mice (10). Therefore,
the unperturbed capacity of FcR-/- mice to
respond to IgE/Ag complexes (Fig. 1
, D and H)
suggests that impaired Ab responses in these animals are seen primarily
after immunization with IgG/Ag complexes. These data provide the first
direct evidence that the ability of IgG1 and IgG2a to enhance in vivo
Ab responses requires functional FcRs.
IgG-mediated enhancement of Ab responses in
FcRIII-/- mice
Previous studies of mice selectively lacking FcRIII
demonstrated an important role of this receptor in inflammatory and
anaphylactic responses and suggested that IgG1 complexes carry out
their effector functions predominantly via FcRIII (16, 26). To our knowledge, no reports regarding the Ab response in
these mice have been published. FcRIII-/-
mice were immunized with BSA-TNP complexed to TNP-specific IgG1, IgG2b,
or IgG2a. As shown in Table I (expts. 1
and 2), all isotypes were able to efficiently up-regulate the Ab
response. When the Ab levels (ng/ml) were compared, the response in
FcRIII-/- animals immunized with IgG2b/Ag
(expt. 1) or IgG2a/Ag (expt. 2) was not markedly different from the
response in wt animals; the response to IgG1/Ag (expt. 1) was lower in
FcRIII-/- mice compared with wt mice. The
differences in SI between the strains in expt. 1 can be explained by
the fact that wt animals had a higher response to BSA-TNP alone than
did FcRIII-/- mice. We conclude from these
experiments that mice lacking FcRIII respond well to IgG/Ag
complexes, and that the magnitude of the response is approximately the
same as that seen in wt animals. To elucidate whether minor differences
exist, more extensive studies need to be performed.
|
RII-/- mice
FcRII contains an immunoreceptor tyrosine-based
inhibitory motif (ITIM) that inhibits activating signals in vitro
through receptors containing ITAMs, such as the B cell Ag receptor
(BCR), TCR, FcRI, and human FcRIIA (reviewed in Ref.
27). FcRII also inhibits BCR-mediated endocytosis
(28) and FcRIIA-mediated phagocytosis
(29). FcRII is present in several isoforms, differing
only in their cytoplasmic tails. The FcRIIB2 isoform, which is
expressed primarily on macrophages, is capable of endocytosing IgG/Ag
complexes (30), whereas FcRIIB1 is expressed on B cells
and contains a sequence that inhibits endocytosis (30, 31). Both isoforms contain ITIM motifs and are capable of
inhibiting cell activation (31).
FcRII-/- mice were shown to produce
5-fold higher Ab titers than wt mice after immunization with KLH-TNP
(in adjuvant) or SRBC (18). To study the role of FcRII
in IgG-mediated enhancement of Ab responses to soluble protein Ags,
FcRII-/- and wt mice were immunized with
BSA-TNP alone or complexed with IgG or IgE anti-TNP. As expected,
the response to IgG/Ag or IgE/Ag in wt mice was significantly higher
than the response to Ag alone (Table I, expt. 3). In
FcRII-/- mice, IgG/Ag complexes were not
only able to enhance Ab responses, but did so much more efficiently
(36- to 189-fold, calculated from the nanogram per milliliter levels of
IgG) than in wt animals. In contrast, the response to IgE/Ag was only
marginally higher (2.4-fold) in FcRII-/-
mice compared with wt mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice. The
fact that FcR-/- mice do not express
functional FcRI or FcRIII (17) suggests that the
binding of IgG/Ag to one or both of these receptors is of primary
importance for the ability of IgG to up-regulate primary Ab responses
to soluble Ag. Assuming that the FcRII in
FcR-/- mice operates normally, the low
response to IgG/Ag in this strain leads to the conclusion that FcRII
is not capable of inducing significant IgG-mediated enhancement; this
interpretation is supported by the strong responses to IgG/Ag in
FcRII-/- mice. The fact that
FcRIII-/- mice respond well to IgG/Ag leads
to the conclusion that FcRI is sufficient for the response to
IgG/Ag. It could be the only receptor involved, although it cannot be
excluded that FcRIII acts in concert with FcRI. Involvement of
FcRI is not surprising, given that targeting of Ag to human FcRI
leads to increased Ag presentation in vitro (33, 34) and
to higher Ab production in mice transgenic for human FcRI
(35). Although FcRI only binds IgG2a with high
affinity, it is possible that other IgG isotypes in complex with Ag are
also captured. Interestingly, when the third extracellular domain of
FcRI is removed, the receptor gains the capacity to bind IgG1 and
IgG2b (36), and a unique allele of FcRI in nonobese
diabetic mice binds IgG2b with high affinity (37). There
is a small residual enhancement in FcR-/-
mice. This may be due to a minor contribution of the C system and
agrees with previous findings that non-C-activating IgG was able to
enhance Ab responses, but that the C-activating wt IgG was slightly
more effective (8). Because the FcR chain is not
exclusive for FcRs, but is also present in the TCR, the lack of Ab
response after immunization with IgG/Ag could hypothetically be due to
a lack of proper T cell help. However, we find this possibility
unlikely, because the response to IgE/Ag complexes (which is
independent of FcRs, but presumably needs the same T cell help as
responses to IgG/Ag) is at least as efficient in
FcR-/- mice as in wt mice (Fig. 1, D and H). In vitro, IgG/Ag complexes are
efficiently taken up via FcR-mediated endocytosis by macrophages or
dendritic cells and presented to Th cells (38, 39, 40). It is
an attractive possibility that this mechanism, operating in vivo,
results in an IgG-mediated enhancement of Ab responses. This means of
inducing Ab responses could be of particular importance in the
induction of secondary immune responses when specific IgG, generated
during priming, is already present at the time of Ag encounter.
Our second major finding is the demonstration of a negative regulatory
role of FcRII in the response to IgG/Ag (Table I, expt. 3). The
response to IgG/Ag in FcRII-/- mice is much
higher than in wt controls, and the FcRII-mediated "suppression"
seen in this system seems to be even more striking than the inhibitory
effect on responses to SRBC or KLH-TNP administered in CFA
(18). The most straightforward mechanism behind the
inhibition of responses to IgG/Ag is that the complexes co-cross-link
the BCR and FcRII, thereby inhibiting optimal B cell signaling after
recognition of Ag. Such negative regulation of B cell activation has
been well documented in vitro (31, 41). An interesting
possibility is that inhibition also takes place at an earlier step in
the chain of events leading to Ab production. Co-cross-linking of
FcRII and the FcR capturing and internalizing the IgG/Ag
complexes (presumably FcRI (and FcRIII), both containing ITAMs)
may inhibit efficient presentation to T cells.
|
Acknowledgments |
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-/- and
FcRII-/- founder animals, Dr. M. Wabl for
IgE, and Dr. P. Coulie for IgG hybridomas. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Birgitta Heyman, Department of Genetics and Pathology, Unit of Pathology, Uppsala University, S-751 85 Uppsala, Sweden. E-mail address:
3 Abbreviations used in this paper: Rh, rhesus; TNP, 2,4,6-trinitrophenyl; KLH, keyhole limpet hemocyanin; FcR, Fc receptor; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif;CR, B cell Ag receptor; SI, stimulation index; wt, wild type.
Received for publication March 5, 1999. Accepted for publication April 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-receptor-deficient mice. Proc. Natl. Acad. Sci. USA 96:2244.RIII (CD16) deficient mice. Immunity 5:181.[Medline]
chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[Medline]
RII-deficient mice. Nature 379:346.[Medline]
-chain does not alter maturation of the B cell response. J. Immunol. 159:2116.RII and FcRIII. J. Exp. Med. 176:469.RIII (CD16). J. Immunol. 161:3026.RII-B1 regulates the presentation of B cell receptor-bound antigens. J. Immunol. 161:2079. receptor-mediated phagocytosis by a nonphagocytic Fc receptor. Blood 91:1762. receptor (monocyte/macrophage)-specific immunogens. J. Immunol. 149:3477.[Abstract]
RI-targeted fusion proteins result in efficient presentation by human monocytes of antigenic and antagonist T cell epitopes. J. Clin. Invest. 98:2001.[Medline]
RI/CD64 triggers enhanced antibody responses in transgenic mice. J. Clin. Invest. 97:331.[Medline]
RI of NOD mice: implications for the evolution of the Ig superfamily. EMBO J. 17:3850.[Medline]
. J. Exp. Med. 179:1109.RIIB modulates B-cell receptor signalling. Nature 369:340.[Medline]
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)
and 20 µg of OVA alone or in combination with 50 µg of TNP-specific
IgG1 (A and E), IgG2a (B
and F), IgG2b (C and G),
or IgE (D and H) (
). Sera taken from
the mice at 14, 21, and 28 days postimmunization were tested for
BSA-specific IgG by ELISA; the geometrical means in micrograms per
milliliter are shown. The level of BSA-specific IgG in normal mouse
serum (CBA/J) was 0.13 µg/ml. This experiment was repeated by
injecting FcR