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*
Department of Immunology,
Medarex Europe,
Genmab, and
Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, University Medical Center Utrecht, Utrecht, The Netherlands;
¶ Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada;
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Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands;
#
Laboratory of Immunohistochemistry and Immunopathology (LIIPAT), The National Hospital, University of Oslo, Oslo, Norway; and
**
Division d’Immunologie et d’Allergie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IgG can initiate a plethora of effector functions, varying from
activation of humoral immune functions by interacting with C1q and
engagement of the classical complement pathway to initiation of
cellular effector functions, including phagocytosis, Ab-dependent
cell-mediated cytotoxicity, and respiratory burst. In contrast, IgA
does not bind C1q and thus cannot activate the classical complement
pathway, whereas IgM is extremely potent in activation of complement
but does not interact with phagocytic cells. Opsonized microorganisms
interact with complement receptors
(CR)3 and Ig-specific
FcR (8, 9), which are present on most myeloid cells. In
humans, one phagocyte IgA receptor, Fc
RI (CD89), has been cloned and
characterized (10), whereas the IgG receptor family
consists of various members. We currently recognize three classes of
leukocyte Fc
R, namely, FcRI (CD64), FcRII (CD32), and
FcRIII (CD16) (8). Polymorphonuclear leukocytes (PMN)
are considered to be crucial effector cells for antibacterial responses
and bear both FcR and CR that mediate activatory signals when
cross-linked. Receptor cross-linking leads to degranulation, formation
of oxygen radicals, and phagocytosis, which may result in destruction
of opsonized microorganisms (9).
The relative contribution of each Ig isotype to host immunity against N. meningitidis has received remarkably little attention. Serogroup B-specific IgG has been shown to initiate complement-mediated lysis of bacteria, as well as leukocyte effector functions such as phagocytosis and respiratory burst (11, 12, 13). Specific IgA has been implicated as a risk factor for disease, because it was shown to impede IgG-mediated bactericidal activity (14), whereas other studies documented its capacity to initiate leukocyte-mediated antimeningococcal activity (15).
Porin (Por) A is a subcapsular protein Ag of group B N.
meningitides that induces Ab formation upon natural infection
(16), and is considered a meningococcal vaccine candidate
(7). To study Ab effector functions to encapsulated
bacteria in detail, we generated a panel of chimeric Abs with identical
mouse variable regions and different human constant regions
(1–4, 1, 2) directed against a single epitope on PorA.
Additionally, secretory IgA (SIgA) molecules were generated by
cotransfecting IgA-producing cells with the human J chain and the
extracellular domain of the human polymeric Ig receptor (secretory
component, SC). Our findings provide new insights into the roles of IgG
and IgA Abs in antibacterial immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The H44/76 strain of N. meningitidis (B:15:P1.7,16)
was stored in Tryptoset broth (Becton Dickinson, Franklin Lakes, NJ) at
-70°C. The day before use, bacteria were plated onto GC agar plates
and incubated overnight at 37°C in 5% CO2.
Bacteria were then subcultured in Trypticase soy broth and grown to OD
0.9 (620 nm). When indicated, bacteria were heat killed at 56°C
(30 min) after they were washed in PBS supplemented with 1% BSA
(fraction V; Boehringer Mannheim, Mannheim, Germany). For phagocytosis
experiments, 1010 heat-killed bacteria in 0.5 ml
PBS were labeled with the pH-stable green dye Alexa 488 (Molecular
Probes, Leiden, The Netherlands). After a washing with PBS (1% BSA),
heat-killed bacteria were aliquoted and stored at -70°C.
Generation of expression vectors
Cloning of genes for the Ab variable regions (VL,
VH) of the mouse IgG2a Ab MN12H2 (17, 18), was performed as described (19, 20). Briefly,
RNA was isolated, and V genes were amplified by RT-PCR, using oligo(T)
primers and avian myeloblastosis virus reverse transcriptase
(Boehringer Mannheim) for cDNA generation and Taq polymerase
(Amplitaq; Perkin-Elmer, Norwalk, CT) and V gene-specific primers for
PCR amplification (18). Variable regions were inserted
into a pUC-HAV20 vector (20), to incorporate the HAVT20
leader sequence (21). After sequence verification using
M13 primers and ABI-Big-Dye-terminator mix (Perkin-Elmer), HAVT20-V
inserts were subcloned into relevant pNUT vectors encoding human
constant regions for the 
-light chain and 1, 2, 3, 4,
1, and 2 heavy chains (20, 22). The panel of Abs
derived from MN12H2 was renamed HMN12H2. Vectors used for the
expression of human J chain and human secretory component (SC) were
described in Ref. 23 .
PCR and sequence reactions were performed in a GeneAmp PCR system 9700 Thermocycler (Perkin-Elmer), and sequence reactions were run and analyzed using an ABI 373 Stretch automated sequencing machine (Applied Biosystems, Foster City, CA).
Ig expression
Heavy and light chain pNUT expression vectors were cotransfected into BHK-21 cells by calcium phosphate precipitation. Positive cells were selected in 1 µM methotrexate (Emthexate PF; Pharmochemie, Haarlem, The Netherlands). Subsequently, IgA-producing baby hamster kidney (BHK) cells were transfected with a J-chain expression vector (IgA-J (23)) and selected using 100 µg/ml hygromycin B (Life Technologies, Gaithersburg, MD). To generate SIgA, the IgA-J-producing cells were additionally transfected with a SC expression vector (IgA-J-SC (23)) and selected using gradually increasing concentrations of histidinol (Sigma, St. Louis, MO; final concentration, 7 mM). For Ab production, cells were grown in roller bottles (Costar, Cambridge, MA) using IMDM (Life Technologies) supplemented with 7.5% heat-inactivated HyClone Fetal Clone1 serum (Greiner, Alphen aan den Rijn, The Netherlands) and 100 µM ZnCl2 to increase production (24).
Ig purification
Supernatants from Ig-producing BHK cells were concentrated using a Prep/Scale TFF concentrator (Millipore, Bedford, MA). IgG supernatants were isolated by affinity chromatography using Sepharose-coupled protein G (Pharmacia, Piscataway, NJ). IgA and IgM were isolated from supernatants using Affi-T columns (BIOZyme, Landgraaf, The Netherlands) (25). Purified Abs were submitted to electrophoresis on 8–18% SDS gradient polyacrylamide gels (Pharmacia) and stained with Coomassie blue. Different molecular forms of IgA, isolated from IgA1-J-SC-transfected cells, were separated by HPLC on HR200 Superdex size exclusion columns (Pharmacia).
Serological assays
Concentrations of purified HMN12H2 Abs were determined by ELISA
using human IgG myeloma subclass Abs (CLB, Amsterdam, The
Netherlands), serum IgA (LC-V Behring Diagnostics, Frankfurt, Germany),
and SIgA (Sigma) as references as standards. Briefly, Maxisorp ELISA
plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with
rabbit anti-human -chain antiserum (Dako, Glostrup, Denmark).
Subsequently, controls (1 µg/ml) and samples were serially diluted in
ELISA plates. For detection, alkaline phosphatase-conjugated rabbit
anti-human IgG, IgA, and IgM serum (Dako) was used. Reactions were
developed with p-nitrophenyl phosphate in diethanolamine
buffer (Sigma), and OD values were obtained at 405 nm. Ab
concentrations were calculated from standard curves.
To confirm the isotypes of HMN12H2 Abs and levels of opsonization, 108 heat-killed bacteria in 10-µl volumes were incubated with 5 µl of 10 µg/ml Abs for 30 min at 4°C, washed twice, and incubated with FITC-labeled Abs to IgG1, IgG2, IgG3, IgG4, IgA1, SC (unlabeled) (Sigma) or anti-IgA (Cappel, West Chester, PA). After a washing, the resulting fluorescence of 104 bacteria was determined using FACS analyses on a FACSCalibur (Becton Dickinson).
Binding experiments
For determination of the relative affinities of chimeric human
Abs, Maxisorp plates were coated with heat-killed bacteria
(109 CFU/ml, 2 h at room temperature), and
the reactivity of HMN12H2 isotypes was examined by incubating Abs over
a broad concentration range. Abs were detected using FITC-labeled
anti- Abs (Southern Biotechnology Associates, Birmingham, AL),
followed by peroxidase-labeled anti-FITC Abs (Boehringer Mannheim)
or alkaline phosphatase-conjugated rabbit anti-human IgG, IgA, and
IgM (Dako).
Bactericidal activity
Serum-mediated lysis of meningococci was determined by serially
diluting chimeric Abs in 96-well V-bottom plates (Greiner) using 100
CFU H44/76 group B meningococci per well, with 12.5% human serum as a
source of complement. The serum was obtained from an individual without
detectable H44/76 meningococcal group B Abs. Plates were incubated for
30 min, and 7-µl volumes (30 CFU) were transferred to GC agar
plates (Oxoid, Basingstoke, U.K.) and incubated overnight. All
incubations were performed at 37°C, 5% CO2,
and 95% humidity.
Phagocytosis assays
Human neutrophils (PMN) were isolated by using Ficoll
(Pharmacia)-Histopaque (Sigma) gradients, followed by hypotonic lysis
of residual RBC in water (for 30 s at 4°C). Abs were serially
diluted in 1-ml polypropylene tubes (Micronics, Lelystad, The
Netherlands). PMN (105) were added along with
5 x 106 Alexa 488-labeled bacteria (see
above) in final volumes of 100 µl and incubated at 37°C for 30 min.
After a washing, samples were resuspended in 300 µl FACS buffer (PBS,
supplemented with 1% BSA and 0.1% azide), and fluorescence
intensities of PMN were measured by flow cytometry. Ig concentrations
were adjusted to 1 µg/ml in assays using fractionated IgA. For all
experiments, cells from FcRIIa-H/H131, FcRIIIb-NA1/NA2
individuals (PCR-allotyped) were used (26).
For quantification of bacteria associated with PMN (phagocytic index),
the same FL1 FACS settings used for measurements of PMN samples, were
also used to measure fluorescence intensities of bacteria, and numbers
of bacteria/PMN were calculated as follows:
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Respiratory burst
Meningococci (107, heat-killed or alive) were preopsonized with the recombinant Abs in volumes of 50 µl for 30 min at 37°C in IMDM supplemented with 1% FCS. When human serum (12.5%, without detectable H44/76 Abs) was used as a source of complement, it was included for the last 5 min of incubation. Tubes were then transferred to a luminometer (Autolumat LB 953; Berthold, Germany), in which chemiluminescence responses were measured every min for 45 min after injection of 100 µl 106/ml PMN and 600 µl 180 µM luminol solution (Sigma) (27).
Statistical analyses
For comparison between total fluorescence of PMN after phagocytosis of fluorescent bacteria, we used Student’s t tests after testing for equal variance. Significance was accepted at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The variable regions of a well-characterized Ab (MN12H2)
(17, 18) directed against the class 1 outer membrane
protein PorA of group B meningococci were cloned. The light chain
variable region (VL) was joined with a human
constant region, and the heavy chain variable region
(VH) with different human heavy chain constant
regions (1–4, 1, 2). The identity of all meningococcal Abs
was verified by sequencing (GenBank accession numbers G5542523
(VL), G5542524 (VH),
AF237583 (1), AF237584 (2), AF237585 (3), and AF237586
(4)). VL- and VH heavy chain
vectors were cotransfected into BHK cells, with or without plasmids
coding for the human J chain, and/or human SC for IgA (IgA-J or
IgA-J-SC). After affinity purification, Abs were assessed for correct
molecular assembly and size. The concentration of whole Abs were
determined in anti-/anti-Fc-AP sandwich ELISA using
well-characterized human myeloma Abs as references.
Ag recognition by chimeric Abs
The characteristics of both recombinant IgG and IgA molecules were
similar to those of reference Abs (Fig. 1
). As can be seen in Fig. 1B
(and data not shown), IgA-J-SC-producing cells produced not only SIgA
but also monomers, indicating, as has been described previously
(23, 28), that single-cell assembly of SIgA is not 100%
efficient. Because this resembles the situation in human serum, we used
total IgA (mIgA, with some dIgA) preparations in the remainder of the
study, unless otherwise stated. Isotypes were verified by
isotype-specific mAbs, in both ELISA and flow cytometric analyses (data
not shown). Binding of chimeric Abs to meningococci could be blocked by
the parental mouse IgG2a monoclonal and by each of the IgG and IgA
chimeric Abs (data not shown). All chimeric Abs recognized the P1.16
epitope of PorA (17) as determined in ELISA by binding to
peptides representing this epitope (data not shown). Binding curves of
IgG and IgA chimeric Abs to whole bacteria, using anti- Abs for
detection, were indistinguishable (Fig. 1, C and
D).
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We studied humoral effector functions of the Ab panel by assessing
their abilities to kill bacteria via activation of human complement.
Human serum with undetectable Ig levels to H44/76 meningococci
(determined by ELISA) was the source of complement. The parental mouse
IgG2a was highly active in this assay (Fig. 2A). Human IgG3 and IgG1
exhibited similar activity, whereas IgG2 had approximately one-tenth
activity. At a concentration of 0.5 µg/ml, HMN12H2 IgG3 and IgG1
triggered killing of 90% of the bacteria. Human IgG4 did not show
detectable bactericidal activity. Heat inactivation of serum abolished
Ab induced bactericidal activity (data not shown). In accord with
previously reported data (14), neither IgA1 nor IgA2
mediated bacterial killing via complement, but both blocked
IgG-mediated killing in a concentration-dependent manner (Fig. 2B). We found that 16 µg/ml IgA2 was sufficient to
completely block IgG1-mediated lysis up to a concentration of 1.5
µg/ml. Higher concentrations of IgG1 overcame this blocking
effect.
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We studied phagocytosis of heat-killed meningococci by freshly
isolated human PMN with each of the IgG and IgA Abs. Human IgG1- and
IgG3-mediated efficient phagocytosis, whereas IgG2 and IgA triggered
lower levels of bacterial uptake (Fig. 2
C). Human IgG4
exerted minimal activity. To confirm that the activity measured in our
assays represented true phagocytosis, internalization was determined by
comparing cells incubated with opsonized Alexa488-labeled bacteria
after incubation at 4°C (attachment) or 37°C (phagocytosis), using
PE-labeled anti-human- antiserum (29) (data not
shown). Because PMN express an IgG receptor (FcRIIa, CD32) that
interacts with human IgG2 and IgG3 in a polymorphic way
(26), we studied PMN from both allotypes. Interaction of
human IgG2-opsonized N. meningitidis was lower for
FcRIIa-R/R131 than IIa-H/H131 and similar, albeit smaller,
differences were observed for IgG3 (data not shown).
Respiratory burst
PMN respiratory burst activity was evaluated with live
meningococci, chimeric IgG and IgA Abs and complement. In Fig. 3A, it can be seen that, in
the presence of complement, IgG1 and IgG3 potently stimulated
respiratory burst, whereas IgG2 was less active. The activity mediated
by IgG1 and IgG3 was highly up-regulated by complement (2-fold),
whereas the IgG2 activity was only moderately enhanced (data not
shown). Human IgG4 exhibited minimal activity in this assay.
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2 µg/ml) Ab
concentrations, IgA stimulated respiratory burst as efficiently as
IgG3, with somewhat delayed responses at higher concentrations (
20
µg/ml) (Fig. 3B). Unlike IgG-triggered respiratory burst,
the activity of IgA-opsonized meningococci was not markedly enhanced by
complement (data not shown).
Interaction of mIgA, dIgA, pIgA, and SIgA with FcRI on PMN
IgA exists as several distinct molecular forms in vivo, mIgA,
dIgA, pIgA, and SIgA (30). Dimeric J-chain-containing IgA
is normally associated with SC on mucosal surfaces. To compare
phagocytic activity of the different molecular forms, we fractionated
IgA1-J-SC on HPLC columns to separate monomeric (fractions 48–51) and
dimeric SC-containing forms (fractions 36–41) (Fig. 1B)
(23). IgA2-J was also fractionated to separate mIgA2,
dIgA2, and pIgA2 from each other. Phagocytosis of heat-killed
meningococci with mIgA1 (Fig. 4A) resulted in significant
phagocytosis (p < 0.001 compared with
background), and to a similar extent as with unfractionated IgA1-J and
IgA2-J (Figs. 4A and 2C). Similarly, mIgA2,
dIgA2, and pIgA2 stimulated phagocytosis (Fig. 4B) and
respiratory burst (data not shown). Remarkably, no phagocytic activity
was found using SIgA1 (Fig. 4A). These data indicate SC of
SIgA to block interaction of IgA with FcRI on PMN.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This work confirmed some of the IgG effector functions documented
before (12, 31). It also yielded data that shed new light
on the impact of IgA-mediated effector functions against encapsulated
bacteria. First, we demonstrated our panel of chimeric Abs to exhibit
correct molecular structure and assembly (Fig. 1) and to bind with
similar affinity (Fig. 1, C and D) to the P1.16
epitope located in surface loop 4 of PorA serosubtype P1.7,16
(17, 18). Our IgA preparations (a portion of which has
dimeric/polymeric properties) exhibited similar binding curves to whole
bacteria as IgG (Fig. 1, C and D), which is
probably explained by the fact that most IgA was monomeric (Fig. 1B). We determined the order of activity of IgG subclasses
to activate human complement to be IgG1 = IgG3>IgG2>>IgG4 (Fig. 2A) (12, 31). IgA Abs have been reported
capable of activating the alternative complement pathway
(32). As previously described (14), however,
IgA Abs did not mediate bactericidal activity and furthermore blocked
IgG-mediated complement activation, likely by interfering with IgG
access to PorA. An alternative explanation may be that IgA binding
resulted in reduced accessibility of C1q to IgG Fc tails by steric
hindrance (33, 34).
We showed that the relative ability of IgG Abs to trigger phagocytic
activation (IgG3>IgG1>IgG2>>IgG4) correlated with predictions based
on knowledge on FcR expression levels (9, 35) and their
relative affinities for IgG subclasses (36). The
relatively low number of receptors for IgG2 (FcRIIa; 30–60,000
receptors/PMN), IgG4 (FcRIa; <300 receptors/PMN), and IgA (FcRI;
6–7,000 receptors/PMN) readily explains the relatively low phagocytic
activities of IgG2, IgA, and IgG4. In addition, the potent capacity of
IgG1 and IgG3 Abs correlates with expression levels and binding
specificity of FcRIIIb 100–300(100–300,000 receptors/PMN), as well as
FcRIIa.
FcR cross-linking reflects to some extent their ability to activate
intracellular signaling cascades, leading to phagocytosis,
degranulation, and eventual killing of microorganisms. We quantitated
PMN activation by measuring the ability of opsonized live meningococci
to stimulate PMN respiratory burst. The chimeric IgG subclasses
stimulated PMN in a similar order as observed in phagocytosis (Fig. 3A). Additionally, we found a major increase in the IgG3 and
IgG1 responses (and a minor increase for IgG2) in the presence of
complement. This effect was absent for IgG4 and IgA Abs (data not
shown). IgA that showed only moderate phagocytic activity and
stimulated respiratory burst effectively. At lower concentrations (2
µg/ml), IgA induced a respiratory burst similar to those of IgG3 Abs
(Fig. 3B). At higher concentrations (20 µg/ml), IgA
still induced an efficient respiratory burst, albeit with a slower
response time than IgG3. This is likely a reflection of lower FcRI
expression levels, compared with those of IgG receptors on PMN
(8, 9). These results are in agreement with those of
others reporting IgA to be more potent than IgG in initiating PMN
respiratory burst (10, 15, 37).
At mucosal surfaces, J-chain-containing dimeric IgA is associated with
the extracellular part of the polymeric Ig receptor (SC), where an
80 Kd SC covalently associates with
dimeric IgA (28, 30). The "docking site" on IgA for
FcRI has been mapped to the boundary of CH2 and CH3 (38, 39), a location occupied by SC (40). To
evaluate the effect of SC on IgA-FcRI binding, we fractionated IgA1
isolated from supernatants of IgA1-J-SC and that of IgA2-J-transfected
BHK cells. In this way, we were able to isolate mIgA, dIgA, pIgA, and
dimeric IgA containing SC (SIgA; Figs. 1B and 4). When
incubated with heat-killed meningococci and PMN, all molecular forms of
IgA facilitated phagocytosis and respiratory burst, except SIgA (Fig. 4), supporting the notion that PMN do not have a receptor for SIgA.
These results agree well with other studies performed in our
laboratory, where various microorganisms were opsonized with
commercially available polyclonal human serum IgA or milk derived SIgA
(41). We extend these studies now by showing that not only
mIgA2 but also dIgA2 and pIgA2 that can stimulate PMN functions,
demonstrating that it is not the dimeric or polymeric nature of the IgA
that is responsible for the lack of binding to CD89, but SC. However,
the observation that SIgA does not interact with FcRI is at variance
with experiments performed with IgA-coated surfaces where both mIgA and
SIgA were capable of initiating PMN respiratory burst
(37). Together, these data support that FcRI interacts
solely with SIgA when coated on plastic or glass surfaces, but not when
used as opsonin on the surface on invading pathogens. Thus, SIgA, which
serves as a "first-line" defense molecule, seems to have
antiinflammatory properties, whereas serum and subepithelial IgA can
interact with human PMN (via FcRI) to stimulate potent
responses.
In summary, we generated a unique panel of human IgG and IgA chimeric
Abs that recognize PorA, a promising vaccine candidate for group B
meningococci. We characterized their capacity to initiate humoral and
cellular effector functions. IgG initiated efficient lysis of
meningococci via complement that furthermore enhanced PMN effector
functions. IgA did not mediate complement-dependent bactericidal
activity and blocked IgG-mediated complement lysis effectively. Despite
a moderate phagocytic capacity, IgA proved most potent in stimulating
PMN respiratory burst. Comparison of IgA1 and IgA2 effector functions
revealed them to be equally effective using heat-killed
Neisseria. However, further studies are needed to evaluate
the influence of IgA1 protease expressed by live bacteria
(42). Remarkably, whereas mIgA, dIgA, and pIgA actively
stimulated PMN functions, SIgA was unable to activate phagocytosis via
FcRI on PMN.
This study established the existence of several differences between IgG and IgA Abs and uncovered an unanticipated level of complexity in the biology of these Abs that are relevant for our understanding of antibacterial immune defense.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jan G. J. van de Winkel, Immunotherapy Laboratory, Department of Immunology, Room KC.02-085.2, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands.
3 Abbreviations used in this paper: CR, complement receptor; Por, porin; BHK, baby hamster kidney cells; MN12H2, mouse mAb directed to PorA from serogroup B (H44/76); HMN12H2, chimeric MN12H2 Abs, with mouse V genes and human constant regions; mIgA, monomeric IgA; dIgA, dimeric IgA; pIgA, polymeric IgA; SIgA, secretory IgA; SC, secretory component; IgA-J, IgA from supernatants of cells transfected with IgA (heavy and light chains) and human J chain; IgA-J-SC, IgA from supernatants of cells transfected with IgA (heavy and light chains), human J chain, and human SC; PMN, polymorphonuclear leukocytes.
Received for publication December 8, 2000. Accepted for publication March 13, 2001.
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References |
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, Fractions that were pooled and concentrated for subsequent
experiments (SIgA, fractions 36–41; mIgA, fractions 48–51).
C and D, H44/76 meningococci-coated ELISA
plates were incubated for 30 min with chimeric Abs over a wide
concentration range. Abs were detected with alkaline
phosphatase-conjugated anti-IgG (C) or FITC-labeled
anti-
p > 0.01; **,
0.01