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,
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Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka, Japan;
Nara Institute of Science and Technology, Ikoma, Nara, Japan;
Organization for Pharmaceutical Safety and Research, Tokyo, Japan;
§
Department of Microbiology, University of Washington School of Medicine, Seattle, WA 98195; and
¶
Department of Neurovirology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) by cross-linking their CD46. The
intracellular events responsible for these immune responses, however,
remain unknown. In this study, we found that 6- to 8-day GM-CSF-treated
peripheral blood monocytes acquired the capacity to recruit
protein-tyrosine phosphatase SHP-1 to their CD46 and concomitantly were
able to produce IL-12 p40 and NO. These responses were induced by
stimulation with mAbs F(ab')2 against CD46 that block MV
binding or by a wild-type MV strain Kohno MV strain (KO; UV treated or
untreated) that was reported to induce early phase CD46
down-regulation. Direct ligation of CD46 by these reagents, but not
intracellular MV replication, was required for these cellular
responses. Interestingly, the KO strain failed to replicate in the 6-
to 8-day GM-CSF-cultured M, while other MV strains replicated to
form syncytia under the same conditions. When stimulated with the KO
strain, rapid and transient dissociation of SHP-1 from CD46 was
observed. These and previous results provide strong evidence that CD46
serves as a signal modulatory molecule and that the properties of
ligands determine suppression or activation of an innate immune system
at a specific maturation stage of human M. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The extracellular portion of CD46 consists of four SCRs and a serine/threonine-rich (ST) domain (15, 16). The intracellular domain is made up of a transmembrane region and a cytoplasmic tail (CYT). Polymorphism of the ST, transmembrane region, and CYT domains, but not SCRs results in multiple isoforms of CD46 (17, 18). The composition of ST domains affects the degree of O-glycosylation, which controls efficacy of ligand binding to the SCR domains (19). CD46 functions were blocked with the mAbs, M75 and M177, which recognize the SCR2 region (11, 12). Since the protein polymorphism of CD46 is caused by alternative mRNA splicing, intracellular domains of each isoform possess different primary structures (16, 17, 18). However, no physiological significance had been suggested for the intracellular heterogeneity of human CD46.
Recent studies indicate that CD46 may possess signaling functions that
modulate cellular responses. Cross-linking CD46 on human macrophages
(M) with either MV, dimerized C3b, or CD46-specific mAbs leads to
inhibition of IL-12 production in response to LPS or
Staphylococcus aureus Cowan (20). Cross-linking
CD46 on human astrocytoma cells with a CD46-specific Ab increases
production of IL-6 (21). Cross-linking of CD46 synergizes
with IL-4 to enhance IgE class switching in the human Ramos B cell line
(22). In human dendritic cells (DC), MV causes increased
expression of costimulators and IL-12, but results in a failure to
induce lymphocyte proliferation (23). Binding of
Neisseria gonorrhoeae to human epithelial cells induces a
transient increase in intracellular calcium (14). These
results suggest that ligand binding to the consensus SCRs of CD46
results in intracellular signaling, which is propagated through
CYT.
In fact, Hirano et al. (24, 25) showed that mouse
M-like cell lines expressing human CD46 produce higher levels of NO
upon infection by MV in the presence of IFN-
. This response is
dependent upon the CD46 CYT, denoted CYT1. Previous reports suggested
that human M and DC are primary targets for MV to modulate
costimulator levels and IL-12 production (20, 23, 26, 27).
These findings are thought to be connected to the immune suppression
seen after MV infection. It is possible that modulation of immune
responses through CYT of CD46 in M/DC could be a major cause of the
MV-mediated immune suppression.
However, it is known that immune activation is another phenotype seen
with measles (28, 29). The lymphocyte proliferation
markers, soluble IL-2 and IFN-, are increased in the early phase of
MV infection (28, 29). In addition, soluble CD8,
reflecting CTL induction, is elevated >1 wk after the measles rash
(28). These findings can be interpreted to indicate that
CD46 signaling governs more than simple suppression of innate immune
responses in human M/DC. The CYT of CD46 might involve a signaling
pathway related to innate immune activation in M/DC even if immune
suppression is a typical or final phenotype in measles. The molecular
mechanisms connecting structural variations of CD46 CYT with reported
M/DC responses, however, are currently unknown.
Here, we found that the two mAbs that block the MV receptor function of
CD46, and a wild-type MV strain Kohno (KO) lead to the activation of
M. The results demonstrated that cross-linking CD46 on human M
with either the KO strain or F(ab')2 of the mAbs
enhanced IL-12 p40 secretion and/or NO production only at the time
point in the maturation process when intracellular tyrosine phosphatase
SHP-1 is recruited to CD46. Furthermore, these effector responses
accompanied a rapid and transient dissociation of SHP-1 from the tail
of CD46. We propose that CD46 can serve as a signal regulatory molecule
for innate immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human monocytic cell line THP-1, the mouse erythroblastoid cell line MEL, the Chinese hamster ovarian tumor cell line (CHO), and the African green monkey kidney cell line Vero were purchased from American Type Culture Collection (Manassas, VA). Human monocytes were prepared from fresh human blood as described below.
The MV strains (30), Nagahata (NV) and Kohno (KO), were
obtained from the Institute for Microbial Diseases, Osaka University
(Osaka, Japan), and the National Institutes of Health, Japan,
respectively. A CAM vaccine strain was purchased from Tanabe (Tokyo,
Japan). The Edmonston (ED) strain was obtained from A. Hirano. The
wild-type strain PB was isolated in 1998 at Osaka University. KO
(30) could be amplified in Vero cells (expressing Vero
CD46 as the MV receptor). Amplification of KO was completely blocked
with the mAb against CD46 (F(ab')2 of M75)
(30). KO induced severe down-regulation of CD46 within 60
min in CHO cells expressing human CD46, although other strains did not
(30). The known properties and the amino acid conversions
in the KO H protein are shown in Table I
.
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mAbs were obtained as follows: CD4, CD14, CD40, and CD64 from PharMingen (San Diego, CA); CD71 from Dako (Copenhagen, Denmark), and CD80 and CD86 from Ancell (Bayport, MN). CD14 microbeads were obtained from Miltenyi Biotec (Bad Gladbach, Germany); FITC-conjugated goat anti-mouse IgG F(ab')2 was purchased from Cappel (West Chester, PA); HRP-labeled goat anti-rabbit IgG was obtained from Bio-Rad (Hercules, CA); anti-SHP-1, anti-Rac 1, and anti-Cdc42 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-IQGAP was a gift from S. Kuroda (Nara Institute of Science and Technology, Nara, Japan) (31); and nonimmune IgG was purchased from Sigma (St. Louis, MO). mAbs against human CD46, M177, M75 (which recognize the SCR2 of human CD46 and block MV receptor activity of CD46); M160 (which recognizes the SCR3 of human CD46 without blocking MV receptor activity); and CD35 (CR1), 243R, were established in our laboratory (11, 32). F(ab')2 of these mAbs were prepared as described previously (33). Anti-MV-H mAbs that block MV infection were obtained from Research Institute for Microbial Diseases (Osaka University) and a generous gift from D. Gerlier (Université Claude Bernard, Lyon, France) (30).
Isolation of monocytes and their differentiation into M
A monocyte-rich fraction was prepared from 400 ml of CPD-supplemented human blood by methylcellulose sedimentation and the Ficoll-Paque method (Pharmacia Biotech, Uppsala, Sweden) as described previously (34). The monocyte-rich fraction was treated with microbeads bearing anti-CD14 mAb and subjected to a MACS system (Miltenyi Biotec). Monocytes (1–5 x 107) were collected with >90% purity from 400 ml of blood. Cells were pelleted, washed, plated in 10-cm plastic tissue culture dishes (no. 25020; Corning, Corning, NY) at 5 x 106 cells/dish, and cultured for 3–12 days at 37°C in 5% CO2 in 10 ml of RPMI 1640 (Life Technologies, Grand Island, NY) containing 100 U/ml GM-CSF (PeproTech, London, U.K.) and 10% FCS (BioWhittaker, Walkersville, MD). Morphological changes were examined under a microscope (IX-70; Olympus, New Hyde Park, NY) (34).
Determination of IL-12 p40 and p70, and IL-18
A sandwich ELISA was performed to determine levels of IL-12 p40, IL-12 p70 (Genzyme, Cambridge, MA), and IL-18 type 1 (MBL, Nagoya, Japan) according to the manufacturer’s protocol. The level of IL-18 type 2 was determined in our laboratory as previously described (35). The absorbance at 490 nm (A490 nm) was measured with a microplate photometer (MTP-120, Corona Electric, Tokyo, Japan).
Determination of NO
The amount of NO in the culture medium produced by human M
was determined by measuring its end product NO in triplicate using a
fluorometric method for mouse M NO (36) with
modification. Briefly, equal volumes (100 µl) of supernatant of
phenol red-free culture medium and 10 µl of 2,3-diaminonaphthalene
(dissolved in 0.62 N HCl; Calbiochem, La Jolla, CA) were mixed in a
96-well plate at room temperature for 10 min. The reaction was
terminated by the addition of 5 µl of 2.8 N NaOH, and the
fluorescence signal was measured at 365 nm on a spectrophotometer
(BioSpec-1600, Shimazu, Tokyo, Japan). A standard curve was obtained
for each experiment using known concentrations of sodium nitrite.
Flow cytometric analysis
Flow cytometry was performed as described previously (32). The FACSCalibur program was used for measurement of the mean fluorescence intensities and comparative analysis.
Immunoprecipitation and immunoblotting
Immunoprecipitation of CD46 and other proteins (CD4, CD35, etc.) was performed as described previously (33). In some experiments a cross-linker (0.75 mM dithiobis-succinimidyl propionate (DSP) purchased from Pierce, Rockford, IL) was used for linking proteins to CD46. For immunoblotting analysis, cells (2 x 106 to 1 x 107 cells) were washed with PBS (pH 7.4) and solubilized with 100 µl of 1% Triton X-100 containing 137 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, 5 mM NaF, and 1 mM PMSF. After centrifugation (10,000 x g for 10 min), aliquots of the supernatants were subjected to SDS-PAGE under nonreducing or reducing conditions (33). Proteins were transferred onto nitrocellulose membranes. The blots were incubated with 10 ml of 10% of skim milk (Morinaga, Tokyo, Japan) for 1 h at 37°C, followed by addition of 10 µg of mAb. One hour later, the membranes were washed extensively with PBS containing 0.05% Tween 20 and then incubated with HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG Abs (Bio-Rad) for 1 h at 37°C. After thorough washing, proteins were detected with an ECL kit (Amersham).
Determination of phosphatase activities in M
CD46-associated phosphatase activity was determined using p-nitrophenyl phosphate as a substrate. The assay was performed at 30°C for 30 min in 20 µl of reaction mixture (20 mM HEPES (pH 7.4), 1 mM EDTA, 10 mM DTT, 10 mM p-nitrophenyl phosphate, and CD46 immunoprecipitates). The reactions were terminated by adding 100 µl of 1 N NaOH. The reaction product p-nitrophenolate was quantified by measuring absorbance at 405–500 nm.
MV infection assay
The levels of messages for MV-H and -N proteins of the KO strain were determined by quantitative RT-PCR, as KO does not form syncytia in Vero cells or human cells. The amount of MV for other strains was expressed as the multiplicity of infection (moi) and then confirmed by quantitative RT-PCR (see below).
Cells were cultured at 70% confluence in 24-well plates (Corning) for
15 h and were infected with MV at an moi of 0.12, which was
determined through dose-response studies for optimal IL-12 p40
production by M. The syncytia formed were observed 2–8 days
postinfection (p.i.) (37). Cells were photographed under
an Olympus microscope (IX-70) (38).
RT-PCR and quantitative RT-PCR
Total RNA was isolated from either MV-infected or noninfected cells according to a standard protocol using guanidium-HCl and acid-phenol/chloroform (39). cDNA was synthesized with SuperScript II RNase H-Reverse Transcriptase (Life Technologies). The primers for detection of MV-H (upstream, 5'-TCAGTAATGATCTCAGCAACTG-3'; downstream, 5'-TTCAATGGTGCCCCACTCGGGA-3') were designed to amplify a 369-bp segment. As a control for the presence of amplifiable RNA, GAPDH primers were used to amplify a 249-bp segment as previously described (34). Amplified PCR products were analyzed by agarose gel (2.0%) containing ethidium bromide (39).
The quantitative RT-PCR assay was performed essentially according to the manufacturer’s guide book. The upstream primer (5'-GATGACAAGTTGCGAATGGAGA-3') and the downstream primer (5'-GACAAGACCCCGTATGAAGGAA-3') were used for PCR amplification of MV-H messages. Similarly, the upstream primer (5'-ACATTAGCATCTGAACTCGGTATCAC-3') and the downstream primer (5'-TTTTCGCTTTGATCACCGTGTA-3') were used for N protein messages. The TaqMan probes for quantitative analysis for H and N proteins were 5'-CCCGAGTGGGCACCATTGAAGGATAA-3' and 5'-CCGAGGATGCAAGGCTTGTTTCAGA-3', respectively. The 5' ends were labeled with a fluorescein derivative (as a reporter), and the 3' ends were labeled with a rhodamine derivative (as a quencher). Three micrograms of total RNA and 200 U of reverse transcriptase were used for one assay. RT was performed for 2 min at 50°C followed by 10 min at 95°C for activation of AmpliTaq Gold (Takara, Tokyo, Japan). PCR was performed for 50 cycles of denaturation for 15 s at 95°C and annealing and extension for 1 min at 60°C using an ABI PRISM 7700 (PE Biosystems, Foster City, CA).
Expression of MV-H mutants on MEL cells and coculture assay with
M
MV-H and MV-F cDNA of the NV strain placed in pME18s mammalian expression vectors were prepared as described previously (38). Mutations of single nucleotides to generate either V451A or I473L and both were introduced into this NV MV-H-based cDNA by site-directed mutagenesis (QuikChange, Stratagene). These two mutations resulted in the conversion of the H protein sequence of the NV strain to that of the KO strain.
Various rodent cells (39) were transfected with the MV-H cDNA of NV, those with V451A or I473L single mutations or that of KO using Lipofectamine (38). Vector only was used as the control. Cells with high expression levels were screened through FACS Vantage using MV-H mAb as a marker. These cells were additionally transfected with MV-F cDNA in some experiments (38). Cells were cultured for 48 h at 37°C in RPMI 1640/10% FCS. Of the cell lines tested, MEL and CHO cells were found to express MV-H and -F with minimal cell damage.
MEL cells (5 x 106/well) expressing MV-H
and -F or only MV-H were poured over human M (5 x
105/well) prepared as described above and
centrifuged at 600 rpm for 3 min. Cells were cocultured for 24 h
at 37°C in 0.5 ml of phenol red-free RPMI 1640/10% FCS. The levels
of NO in the supernatants were determined as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human monocytes were cultured with GM-CSF to induce
differentiation to M. During the process of maturation, we detected
an intracellular phosphatase SHP-1 coprecipitating with the MV receptor
CD46 on day 7 (Fig. 1A). SHP-1
reproducibly coprecipitated with CD46 during days 6–8. The CD46-SHP-1
association was specific in a certain activation stage of M, since
1) M-CSF (substituted for GM-CSF) did not allow the recruitment of
SHP-1 to CD46 in 7-day cultured M (data not shown); 2) no SHP-1 was
coprecipitated with an Ig superfamily protein CD4 (Fig. 1A);
and 3) the CD46-SHP-1 association was observed in a human M-like
cell line, THP-1 (Fig. 1B). M express another SCR protein
CR1 (CD35) concomitantly with cell differentiation, and CR1 may
coprecipitate SHP-1, notably not during days 6–8 but after day 9 M
(Fig. 1A). The SHP-1 recruitment to CD46 is again specific
to day 6–8 M. CD46 is known to bind the cytoplasmic protein moesin
(40, 41), which is associated with the small G protein Rho
involved in cell motility (41, 42, 43). Other G
protein-related molecules, including Cdc42, Rac1, and IQGAP, failed to
be connected to CD46 in any stage of M (data not shown) or THP-1
even using a cross-linker (Fig. 1B).
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During M differentiation, the cellular responses occurring
through CD46 were investigated. CD46 of monocytes/M was cross-linked
with either F(ab')2 of mAbs M177, M75 (data not
shown), or M160 or with the MV strains ED, NV, PB, CAM, or KO at each
differentiation step. M responses, including IL-12 and NO
production, were analyzed after the addition of these stimulators.
Within 24 h the addition of the wild-type strain KO strongly
induced the production of IL-12 p40 in 6- to 8-day GM-CSF-cultured M
(Fig. 2A). Other strains,
including ED and NV, did not induce IL-12 p40. Similar results were
obtained with mAbs. The F(ab')2 of M177 and M75
preferentially induced IL-12 p40 from the 6- to 8-day GM-CSF-cultured
M, whereas the F(ab')2 of M160 did not (Fig. 2B). Thus, the mAbs that block MV receptor function of CD46
induced similar IL-12 p40 production as the KO strain. IL-12 p70 as
well as either type of IL-18, however, were barely detected under the
conditions tested (data not shown). Other factors produced by M
during maturation appeared not to be involved in IL-12 production,
since changing the medium had no effect on IL-12 levels during each
step of M maturation. Thus, CD46-mediated IL-12 p40 induction
depends on the M maturation step characterized by SHP-1 recruitment
to CD46 and on the ligands with which CD46 is stimulated.
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was assessed in monocytes and day 3, day 7,
and day 12 GM-CSF-cultured M. Unexpectedly, monocytes responded to
the CAM vaccine strain, resulting in the release of a moderate amount
of NO into the culture medium (Fig. 3).
In contrast, virtually no NO was detected at any stage of M
maturation with any MV strain other than KO despite the addition of
IFN-. A high level of NO production was induced again by the KO
strain only in 7-day GM-CSF-cultured M (Fig. 3), although there was
sample-to-sample variation in NO production by M. Interestingly,
this response occurred at the same time as SHP-1 recruitment to CD46.
In contrast, cross-linking CD46 with M177 F(ab')2
did not result in NO production in 7-day GM-CSF-cultured M (data not
shown).
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by MV stimulation
The expression levels of APC-related molecules were next assessed
by flow cytometry. Surface levels of CD14, CD40, CD46, CD71, CD80, and
CD86 on monocytes were compared with those on M cultured with GM-CSF
for 7 days (Table II). Significant
elevations of CD86 and CD80 were detected in 7-day-cultured M after
the addition of KO (underlined in Table II), but not in monocytes. In
comparing day 3, day 6, and day 12 cultured M, the day 6 cultured
M were found to exhibit the most significant alterations (data not
shown), again consistent with the time frame of SHP-1 recruitment
to CD46.
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cellular responses
To rule out the possibility that products, including dsRNA,
resulting from MV replication contribute to IL-12 p40 production,
UV-irradiated MV strains were used to stimulate 7-day GM-CSF-cultured
M. The UV-irradiated KO strain retained the ability to induce IL-12
p40 in M, although somewhat less potently (Fig. 4). The replication ability of MV was
completely abolished by UV treatment. One day p.i., IL-12 was again
undetectable after the addition of another MV strain regardless of UV
irradiation. Thus, direct stimulation of CD46 with KO caused the
induction of IL-12 p40 independently of viral replication. Induction of
IL-12 p40 by direct binding of mAb against CD46 also supports this
interpretation and parallels the previous finding that the receptor for
KO is CD46 (30).
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We then analyzed the phenotypes of M infected with the NV and
KO strains. MV amplification in 7-day GM-CSF-cultured M was
evaluated 4 h to 7 days p.i. (moi = 0.12 or equivalent) using
quantitative RT-PCR. MV-H mRNA was detected at 18 h and peaked at
48 h for the NV strain (Fig. 5).
Similar results were obtained after infection with ED and PB (data not
shown), but not with KO (Fig. 5). The MV-H message for KO was slightly
increased at 24 h, yet decreased at 48 h p.i. and was not
present on day 7 p.i. Even at the maximal time point, the relative
level of viral mRNA was about 500 times lower in KO-infected M than
in M infected with other viral strains. With the KO strain, again
the N protein message of MV failed to be detected in 7-day
GM-CSF-cultured M after 48 h p.i. (data not shown). Unlike
other strains, amplification of KO was severely suppressed in M.
Thus, only the KO strain activates M to induce IL-12 p40 and NO and
to up-regulate surface levels of costimulators, yet, unlike other MV
strains, it failed to replicate or even survive in the targeted
M.
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. Monocytes were cultured for 3 and 6
days with or without GM-CSF, then inoculated with the NV, KO, or ED
strain of MV (moi = 0.12 or its equivalent), and morphological
changes were followed under a phase-shift microscope. M formed
syncytia 2–3 days p.i., while monocytes were highly resistant to
syncytium formation. In Fig. 6, MV-mediated syncytia were observed in
day 3 and day 6 cultured M when either the NV or ED strain was
added. Cells infected with KO would not form any syncytium under
any of these conditions. Instead, KO induced a scattered pattern in
both monocytes and GM-CSF-treated M (Fig. 6, left
column). This tendency was also seen in day 6 cultured M (Fig. 6, right column). GM-CSF accelerated M proliferation and
attachment to the dish (top row, middle
and right columns of Fig. 6). Infection by ED or NV resulted
in cells with similar morphological features, while KO infection
produced elongated cells. Infection with KO through CD46 suppressed
viral amplification and induced a unique phenotype in human M,
rather than typical syncytium formation.
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SHP-1 is involved in the negative regulation of immune responses,
as it is a major phosphatase in lymphocytes and M (44).
As shown in Fig. 7A
(Panels A and B), KO input induced a rapid and
transient dissociation of SHP-1 from CD46 in day 7 cultured M. After
addition of NV (Fig. 7A) and ED (data not shown) strains,
however, a much more gradual reduction was seen in the amount of SHP-1
associated with CD46 over a 30-min period. Repetitive analysis
suggested that SHP-1 dissociates from CD46 15–20 min after KO input in
M. Kinetics of CD46-associated phosphatase activity paralleled the
amounts of CD46-bound SHP-1 (Fig. 7C). Total phosphorylated
proteins were slightly increased concomitantly with dissociation of
SHP-1 in KO-infected M (Fig. 7A, Panel D). In
the human monocytic cell line THP-1, KO induced SHP-1 dissociation from
CD46 within 15 min, with reassociation at 30 min (Fig. 7B).
This early response was not observed in THP-1 cells with NV.
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To confirm that MV-H of KO is responsible for M cellular
responses, we performed coculture studies in which human M were
incubated with MEL transfectants expressing MV-H of a KO-type mutant,
chimeric mutants (both generated from MV-H of NV), or intact MV-H of NV
(Fig. 8). The construct of KO-type MV-H
was made by the addition of two mutations to the construct of MV-H of
NV (which conferred the same amino acid sequence as KO MV-H)
(30). MEL cells expressing MV-H with the two mutations
potentiated NO production by human M. Other mutant- and intact NV
H-expressing cells barely induced augmentation of NO production by
M. Similar results were obtained with cells expressing both H and F.
Although the background NO levels were high, presumably due to species
difference between M and stimulant cells, significant enhancement of
NO production was reproducibly observed by stimulation with KO-type H
protein. A similar tendency was observed with IL-12 p40 (data not
shown). Virtually, no enhancement of NO and IL-12 production was
observed with monocytes and <3-day GM-CSF-cultured cells (data not
shown). Hence, the M cellular responses are attributable to the
specific sequence of H protein and the maturation stage of
M.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study demonstrated that CD46 allows signaling in human M. An
intracellular phosphatase, SHP-1, was found to be stage specifically
recruited to CD46 in M. After stimulation with ligand, SHP-1
transiently dissociated from the tail complex of CD46. Interestingly,
these responses were induced in 6- to 8-day GM-CSF-cultured M
stimulated with the wild-type MV strain KO or with the mAbs that block
the MV receptor function of CD46. Examples of cellular responses
propagated by M CD46 include induction of IL-12 p40, NO production,
and up-regulation of the surface levels of costimulators. A scattered
morphology was also induced in M after CD46 cross-linking, similar
to that reported for early phase down-regulation of CD46
(30). These findings suggest that SHP-1, moesin, and
presumably unknown phosphatase substrates are involved in an
intracellular complex with CD46, and that ligand stimulation of CD46
during virus infection plays a major role in signal transmission and
cellular responses. However, the participation of another MV receptor
or other virus-binding molecules (47) in the observed
immune responses cannot be ruled out.
MV infection induces transient suppression of host immunity, leading to secondary infections that are a major cause of death in measles patients (48, 49). MV-infected APCs can suppress the proliferation of unaffected lymphocytes, most likely through cell-to-cell contact (23, 26, 27). Temporal lymphopenia and loss of delayed-type hypersensitivity reaction were found in measles patients, which cannot be fully explained by MV susceptibility of lymphocytes or MV-mediated cytolysis (48, 49). Recently, the mechanism of immune suppression has been investigated in association with CD46 function in APC (20, 23, 26, 27).
The present data suggest, however, that in some cases M are
activated through CD46, as measured by levels of surface markers,
cytokines, and NO production. Our results demonstrating the inability
of the KO strain to grow in the CD46-prestimulated M may in part
agree with the previous idea that MV infection produces low levels of
viral proteins and virtually no infectious virus in M
(37). An attractive hypothesis is that APC serve as
reservoirs of infectious materials and their CD46 CYT may essentially
activate M/DC to eliminate invading materials. Indeed, immune
activation profiles have been observed in the early phase of infection
in measles patients (28, 29). In addition, Schnorr et al.
(23) showed that human DC precursors are matured by
infection with MV, particularly the wild-type WTF strain, resulting in
the up-regulation of HLA-DR, CD83, and CD86 as well as IL-12 synthesis.
Our DC analysis with the KO strain is currently in good agreement with
these reports, supporting the presence of another CD46 functional
pathway, immune activation. Although M and DC have distinct
properties, these results with DC parallel our results with M. MV
strains free from immune suppression would be useful for the
development of measles vaccine and therapeutic vectors.
Three cross-linkers of CD46, including mAbs, C3b dimer, and MV (ED),
are all reported to lead to the suppression of IL-12 production in
human activated M (20). Unlike M75 and M177, other mAbs
tested could not inhibit MV infection (20). The binding
sites on CD46 for M75 and M177 contain Arg69
(11, 50, 51), which is not shared with the epitopes of
other mAbs. The primary structure of the H protein of the KO strain
differs from those of the ED and NV strains (30). The CAM
vaccine strain also possesses a unique H protein (52) that
may cause the unidentified monocyte-stimulating activity (Fig. 3
). We
confirmed that MV strains other than KO and the mAb M160, which did not
block the MV receptor function of CD46, tended to suppress the
stimulation-dependent production of IL-12 p40 (data not shown).
Immune-suppressive signal may be a common feature compared with
activation signal in CD46 cross-linking (20).
One possible explanation for the functioning of CD46 in both the
suppression and activation of cellular responses is the existence of
multiple signaling pathways, involving the various isoforms of CD46
with their differing cytoplasmic tails. Additionally, the properties of
the ligands for CD46 appear to be critical in the activation of M.
The two amino acids Val451 and
Ile473 in the H protein of the NV strain were
converted to Ala451 and
Leu473 in the H protein of the KO strain
(30). These differences are localized within (or near) the
region reported to be involved in H binding to CD46
(53, 54, 55, 56). Thus, an alternative interpretation is that the
occurrence of immune suppression or activation depends on the portion
of CD46 that is accessible to mAbs or ligands. IL-12 regulation, for
example, depends on the properties of the ligands and signaling.
Additionally, our study demonstrates that the ligand-binding portion of
CD46, but not MV replication, is important for determining positive or
negative cellular responses. Other factors, including the maturation
stage of the M, may contribute to the final outcome of the
response.
It is surprising, however, that only a two-amino acid difference in the
H protein can cause opposite responses. The altered amino acids are
near the Tyr481/Asn, which may determine
CD46-mediated down-regulation and CD46 binding capacity
(53, 54, 55). We have made the cDNA constructs encoding the H
protein of KO by site-directed mutagenesis. The relevant point was in
part confirmed by stimulating M with murine cells expressing various
mutant H proteins but no CD46 counterpart.
CD46 was initially identified as a C regulator that was ubiquitously
expressed on human nucleated cells (2, 18) and has not
been analyzed as a signal-transducing receptor. Previous reports
suggest that M produce C3, which, in turn, is activated by M
surface proteases (57, 58). The activated C3b can then
bind back to the M in an autocrine fashion (57). The
C3b binding to CD46 may also be involved with the SHP-1-dependent
regulatory mechanism of M.
We have not yet identified the mechanism of SHP-1 recruitment to CD46.
The presence of two SH2 domains in SHP-1 provides a structural
prerequisite for the diverse range of molecular interactions in which
this phosphotyrosine phosphatase appears to participate
(59). The VxYxxL sequence in the CD46 juxtamembrane domain
(60) may be too close to the inner surface of the plasma
membrane to allow access of SHP-1 to this sequence. There are no
immunoreceptor tyrosine-based inhibitory motif present in any isoform
of the CD46 molecule (61). Thus, it remains unclear
whether the effect is mediated directly by receptor phosphorylation or
indirectly by phosphorylation of receptor-associated proteins or
protein tyrosine kinases. Recently, IL-12 p40 production was reported
to be regulated through the stress-activated protein kinase (SAPK)
activation pathway (62). The family of mitogen-activated
protein kinase kinase proteins is about 40 kDa and may be
phosphorylated during SAPK activation (62). Indeed, our
preliminary data suggest that a CD46-associated 40-kDa protein can be
detected concomitantly with dissociation of SHP-1 from CD46 by in vitro
kinase assay in the 7-day GM-CSF-cultured M only when the M were
treated with the KO strain. Studies of motheaten mice
(me/me), known to be homozygous for loss-of-function
mutations in the SHP-1 gene (63), as well as human CD46
transgenic mice may give some insight into these possibilities.
M/DC are the main effectors of the innate immune system. They
express receptors for foreign material, i.e., bacteria, fungi, and
viruses, and mature into active phenotypes through stimulation with
ligand. The ligands for these receptors are known to contain
pathogen-associated molecular pattern (PAMP) (64, 65).
Recently, representatives of the PAMP receptors were recognized as
Toll-like receptors (66, 67, 68, 69). It has been found that many
proteins with SCR also serve as bacteria and virus receptors
(70). We hypothesize that both SCRs and leucine-rich
repeats in Toll-like receptors are PAMP recognition motifs that
modulate innate immune functions. The responses via these receptors
lead to the activation or suppression of the immune system. In this
study, we show that a SCR protein, CD46, which was previously
identified as a C regulator and an MV receptor, also has a novel signal
regulatory function in M.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tsukasa Seya, Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan.
3 Abbreviations used in this paper: MCP, membrane cofactor protein; CAM, a measles vaccine strain; CYT, cytoplasmic tail; DC, dendritic cells; ED, Edmonston MV strain; KO, Kohno MV strain; MV, measles virus; M, macrophages; moi, multiplicity of infection; NV, Nagahata MV strain; PAMP, pathogen-associated molecular pattern; p.i., postinfection; SAPK, stress-activated protein kinase; SCR, short consensus repeat; SHP-1, a protein-tyrosine phosphatase; ST, serine/threonine-rich domain.
Received for publication January 4, 2000. Accepted for publication July 26, 2000.
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