Endogenous humoral autoreactive immune responsesto apoptotic cells: Effects on phagocytic uptake,chemotactic migration and antigenic spread

Joy Das, Pooja Arora, Donald Gracias, Alagangula Praveen, Beena P. J. Raj,

Elishba Martin and Rahul Pal

Immunoendocrinology Laboratory, National Institute of Immunology, New Delhi, India

Enhanced cell death and deficient clearance of cellular debris are thought to contribute to

increased self-antigen exposure in systemic autoimmune disease. To investigate the

characteristics of early humoral autoimmune responses, six monoclonal antibodies were

generated from two autoimmune prone strains of mice. All antibodies specifically bound

the surface of late-stage apoptotic cells. Similar antibody reactivities were present in the

sera of patients with systemic lupus erythematosus. While IgM antibodies significantly

reduced the phagocytic uptake of apoptotic thymocytes, IgG antibodies enhanced uptake.

Poly-reactivity was demonstrated in the recognition of ribonucleoproteins and lipids. An

antibody reactive towards lysophosphatidylcholine reversed lysophosphatidylcholine-

mediated inhibition of LPS-induced TNF-a production and adversely affected the trans-

migration of phagocytes towards an apoptotic stimulus. In several instances, CDR were

characterized by the accumulation of somatic mutations. Anti-idiotypic antibodies

generated upon immunization bound distinct cellular moieties and self-antigens. Poly-

specific, apoptotic cell-reactive autoantibodies can therefore directly impact upon the

course of disease by influencing phagocytic uptake of apoptotic cells, by inducing a pro-

inflammatory environment through neutralization of bioactive lipids, by blinding phago-

cytes to the presence of dying cells through the negation of lipidic chemotactic signals, and

by mediating diversification of the humoral autoimmune response via the idiotypic

network.

Key words: Apoptosis . Autoimmunity . Systemic lupus erythematosus

Introduction

Systemic lupus erythematosus (SLE) is the prototypical organ

non-specific autoimmune disease. The disease generally mani-

fests a lifelong, relapsing–remitting phenotype. Multiple organs

are targeted; chronic renal failure is a serious consequence, and

patients can also exhibit arthritis, neurological involvement and

immune dysfunction [1]. Genetic pre-disposition has been

described in both animals and humans, and various susceptibility

loci have been identified using genetic studies [2]. Both

spontaneous and derived murine models of disease exist and

have been extensively utilized to study pathology [3].

Despite significant advances in understanding lupus-related

organ dysfunction, drawing correlations of disease manifesta-

tions with autoimmune specificity remains a high priority. More

than 100 different antibody specificities have been described and,

while most may be epiphenomena, some autoantibodies may

have destructive potential. Anti-dsDNA antibodies have been

associated with kidney pathology [4], anti-Ro and anti-La

responses with neonatal lupus and congenital heart block [5],Correspondence: Dr. Rahul Pale-mail: rahul@nii.res.in

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2008. 38: 3561–3574 DOI 10.1002/eji.200838624 Clinical immunology 3561

and anti-phospholipid antibodies with recurrent spontaneous

abortion [6]. Additionally, titers of complement-fixing antibodies

to cell-surface moieties upon leucocytes are negatively correlated

with circulating leucocyte numbers [7]. While some studies

indicate a correlation between anti-ribosomal P antibodies

and neuropsychiatric manifestations [8], contradictory data

have also emerged.

Interestingly, in both SLE patients and several disease-prone

animal models, aberrance in apoptotic processes has been observed.

Several studies document excessive spontaneous apoptosis (or

excessive susceptibility to apoptotic stimuli) in lupus patients [9], as

well as an inefficient clearance of apoptotic debris [10]. Though

apoptosis is considered a non-inflammatory event, infusion of

experimental animals with syngeneic apoptotic cells generates a

lupus-like autoimmune response [11], albeit with no associated

long-term pathology. In furtherance of the postulate-linking

apoptosis and systemic immune dysfunction, animals genetically

modified to impair the uptake of apoptotic cells exhibit lupus

pathology [12, 13]. Apoptotic debris may therefore be the source

of antigenic material that initiates autoreactivity in genetically

susceptible individuals.

Upon apoptosis, cells externalize phosphatidylserine (PS) as

an early event [14]. Subsequently, ‘‘blebs’’ containing both

nuclear and cytoplasmic autoantigens extrude from the cell

surface [15], and these particles quite possibly constitute the

initial immune trigger. Autoantibodies known to target

several lupus-specific antigens bind to the surface of apoptotic

cells before true cellular permeability is achieved [16]. It is

conceivable, therefore, that autoantibodies specifically reactive

to the apoptotic cell surface could have disease-modifying

effects.

This report seeks to investigate properties of the apoptotic

cell-specific antibody responses in lupus in the context of disease.

Apoptotic cells, rather than individual molecules known to be

externalized during apoptosis, served as targets in the

development of six murine monoclonal antibodies (2C11,

IgMk; 2H8, IgMk; 1B4, IgMk; 2C3, IgG2bk; 1B3, IgG2ak; 1B1,

IgG2ak) from two autoimmune-prone strains of mice.

Caspase activity was shown to be critical for antibody binding.

Antibodies exhibited varying degrees of cross-reactivity to

commonly targeted ribonucleoprotein (RNP) autoantigens as

well as phospholipids, and impacted upon the phagocytic uptake

of apoptotic cells in an isotype-dependent manner. Antibody

2C11, which bound lysophosphatidylcholine (LPC), influenced

two important biological roles mediated by the lipid: the

suppression of inflammatory responses and the chemotaxis of

phagocytic cells. Antisera, generated upon immunization with

either antibody 2C11 or antibody 1B1, exhibited anti-self-reac-

tivity with a diversified antigenic recognition profile. Variable

region analysis revealed that distinct gene families were

employed, with some antibodies exhibiting preferential accumu-

lation of somatic mutations in the CDR. Germline or somatically

mutated early antibody responses specifically directed against

dying cells could thus conceivably impact upon the course and

severity of disease.

Results

Reactivity towards intra-cellular and cell-surfaceantigens

Table 1 presents nomenclature, cellular binding pattern, Ig

isotype and animal source of the antibodies.

A total of 465 independent hybridomas were established, using

spleen cells derived from aging, lupus-prone mice. Of these, 32

secreted antibodies reactive to the surface of healthy cells, and 58

secreted antibodies reactive towards permeabilized cells and non-

reactive towards apoptotic cells. Figures 1 and 2 show the binding

properties of six antibodies chosen for their specificity towards

permeabilized as well as apoptotic cells, and non-reactivity towards

moieties present on the surface of healthy cells. Antibodies 2C11 and

1B4 exclusively recognized cytoplasmic moieties while antibodies

1B1 and 1B3 bound nuclear antigens. Antibodies 2H8 and 2C3

bound antigens in both regions (Fig. 1A–F). Similar binding patterns

were observed on many cell types and across two species (humans

and mice), indicating evolutionary and lineage antigenic conserva-

tion (data not shown). Relevant isotype control antibodies did

not bind either permeabilized or non-permeabilized cells (Fig. 1G

and H).

Reactivity towards apoptotic cells

Eight hours after the induction of apoptosis, cells that bound

antibody also bound Annexin-V (Fig. 2A, top right quadrants) and

cells not binding Annexin-V were also not bound by antibody

(Fig. 2A, bottom right quadrants). Cells that bound Annexin-V but

not antibody (Fig. 2A, top left quadrants), as well as healthy calls

that bound neither reagent (Fig. 2A, bottom left quadrants), were

also observed. Addition of the pan-caspase inhibitor z-VAD-fmk to

cultures during the induction of apoptosis caused a reduction in both

Annexin-V and antibody reactivity; the few cells recognized by the

antibodies also bound Annexin-V (Fig. 2B).

Upon confocal microscopy, all six antibodies exclusively

bound cells also recognized by Annexin-V (Fig. 2C), reiterating

results obtained by FACS analysis. While Annexin-V reactivity

appeared to be relatively more evenly distributed across the cell

surface, in most instances antibody reactivity appeared confined

to restricted regions of the membrane, possibly upon incipient

Table 1. Description of nomenclature, intra-cellular specificity, isotypeand animal source of the monoclonal antibodies

Antibody Specificity Isotype Strain

2C11 Cytoplasmic IgMk C57BL/6lpr/lpr

2H8 Cytoplasmic/nuclear IgMk C57BL/6lpr/lpr

1B4 Cytoplasmic IgMk NZB/W F1

2C3 Cytoplasmic/nuclear IgG2bk NZB/W F1

1B3 Nuclear IgG2ak NZB/W F1

1B1 Nuclear IgG2ak NZB/W F1

Eur. J. Immunol. 2008. 38: 3561–3574Joy Das et al.3562

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

blebs. Regions of overlap between Annexin-V and antibody

reactivity appeared yellow or orange in merged images, as in

images depicting Antibodies 1B4 and 2C3. Healthy cells not

binding Annexin-V or antibody (as in the image depicting

Antibody 1B4 reactivity), as well as cells at the early stages

of apoptosis, which bound Annexin-V but not antibody (as in

images depicting antibodies 1B3 and 1B4 reactivity), were

apparent.

At 12 h post-apoptotic stimulus, preferential antibody binding to

surface protrusions (putative apoptotic blebs) was more significant

(Fig. 2D). Antibody 2C11 bound blebs isolated from apoptotic SP2/0

cells (Fig. 2E). In addition, blebs isolated from apoptotic 2C11

hybridoma cells, as opposed to blebs isolated from apoptotic SP2/0

cells, were demonstrated to contain adhered antibody (Fig. 2E).

Antigenic analysis

IgM antibodies appeared to be reactive towards multiple RNP

(Fig. 3A). Antibody 2C11 predominantly bound Ro52 and the

U1-RNP A Protein, while antibody 2H8 bound Ro60 to the most

significant extent, but also demonstrated recognition of Ro52,

SmB, SmD and the U1-RNP A Protein. Antibody 1B4 recognized

Ro52 and Ro60, while demonstrating minor reactivity towards all

the other proteins. Similar (but distinctive) poly-reactivity was

observed with the IgG antibodies (Fig. 3B). Antibodies 2C3 and

1B1 bound Ro60 and SmD in a dominant fashion, while antibody

1B3 exclusively recognized SmD. Antibody 1D1 (IgG3k), which

was generated as part of another study and recognized the cell

membrane of healthy, non-apoptotic Jurkat cells, was employed

as control; it was essentially non-reactive.

Antibodies to oxidized phospholipids have been shown to bind

to the apoptotic cell surface and induce the release of pro-

inflammatory cytokines [17]. The IgM antibody 2C11 specifically

bound LPC and the IgG antibody 2C3 exhibited a variable degree

of cross-reactivity to different lipids. The other antibodies were

poorly reactive (Fig. 3C and D).

Human SLE sera were evaluated to accord clinical relevance

to the data. The autoantigen Ro60 was chosen for this analysis

since five of the six antibodies exhibited appreciable binding to it.

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Figure 1. (A–F) Recognition of permeabilized SP2/O cells (solid, thick profiles) by the IgM (A: 2C11; B: 2H8; C: 1B4) and IgG (D: 2C3; E: 1B3; F: 1B1)monoclonal antibodies. Non-permeabilized cells (dashed profiles) were not recognized. Solid, thin profiles indicate negative controls, stainingobtained in the presence of only secondary antibodies. Also depicted (at right for each antibody) are respective intra-cellular localization patternsupon permeabilized cells on immunofluorescence analysis (top) and corresponding phase contrast images (bottom). (G and H) Binding of isotypecontrol antibodies (IgMk: red profiles; IgG2ak: green profiles; IgG2bk: blue profiles) to (G) permeabilized and (H) non-permeabilized SP2/O cells. Grayprofiles indicate negative controls, staining obtained in the presence of only secondary antibodies.

Eur. J. Immunol. 2008. 38: 3561–3574 Clinical immunology 3563

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Figure 4A depicts the anti-Ro60 reactivity of human SLE sera as

well as an equal number of normal human sera. Competition

analysis was carried out using SLE sera, or normal sera, and

antibody 2C3 (Fig. 4B). Unlike antibodies in normal sera,

antibodies in sera from SLE patients were able to efficiently

compete with the monoclonal antibody for binding to Ro60.

Similar results were obtained for the other antibodies (data not

shown).

Biological analysis

Effects on the phagocytosis of apoptotic cells

All IgG antibodies caused significant increases in the phagocytic

uptake of apoptotic cells (Fig. 5A). In contrast, the IgM antibodies

caused dose-dependent decreases in phagocytic uptake; at the

highest concentration tested, internalization of apoptotic cells

was decreased to ‘‘background’’ levels, as defined by the extent of

uptake observed when healthy cells were employed (Fig. 5B).

The antibodies had no effect upon the phagocytic uptake of

healthy cells (data not shown).

Effects of neutralization of LPC

LPS induced the secretion of TNF-a by murine peritoneal exudate

cells. Addition of LPC to the cultures significantly inhibited

secretion of the cytokine. Antibody 1B4 (employed here as an

LPC non-reactive isotype control) did not affect the LPC-induced

decrease in the production of TNF-a by LPS. In contrast, the

LPC-reactive antibody 2C11 significantly dampened the down-

modulatory effects of LPC (Fig. 6A). Cell viability in all cultures

remained greater than 90%.

The effect of antibody 2C11 on phagocytic migration was then

ascertained. As expected, supernatant derived from apoptotic

cells caused enhanced transmigration of THP-1 cells in compar-

ison with supernatant derived from healthy cells. Significantly,

antibody 2C11 caused dose-dependent diminution in the

chemotactic movement of cells, whereas an isotype control

antibody had no effect (Fig. 6B).

1 375 KDa

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Figure 2. Preferential recognition of Annexin-V positive Jurkat cells by the monoclonal antibodies 8 h after induction of apoptosis. Apoptosis wasinduced in the (A) absence or (B) presence of the pan-caspase inhibitor z-VAD-fmk. (C) Binding of the antibodies to cultures containing apoptoticJurkat cells, as assessed by confocal microscopy. Green: Annexin-V; red: respective antibodies. (D) Preferential localization of antibody 2C11 uponbleb-like projections (arrows) 12 h after the induction of apoptosis. (E) Blebs (5 mg protein) purified from apoptotic SP2/0 cells were incubated withantibody 2C11 and washed. The presence of bleb-associated Ig was then assessed by Western blot (Lane 1). Blebs (5mg protein) isolated from eitherapoptotic SP2/0 cells (Lane 2) or apoptotic 2C11 hybridoma cells (Lane 3) were processed for Western blot to assess the presence of endogenouslyadhered Ig. In Lanes 1 and 3, bands at 75 and 25 kDa correspond to the heavy and light chains, respectively, of adhered antibody 2C11 (IgMk).

Eur. J. Immunol. 2008. 38: 3561–3574Joy Das et al.3564

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Anti-idiotypic responses

Antibody 1B1 (IgG2ak) was immunized in 6 wk-old NZB/W F1

lupus-prone mice, the mouse strain from which the antibody was

derived. As shown in Fig. 7A (right panel), upon FACS analysis,

sera demonstrated enhanced presence of IgG antibodies reactive

towards self-antigen(s), in comparison with sera generated by

immunization with adjuvant alone; antibodies of the IgM isotype

did not exhibit such anti-self-reactivity (Fig. 7A, left panel). As

previously indicated, antibody 1B1 predominantly demonstrated

reactivity towards nuclear antigen(s), with the cytoplasm

remaining unbound (Fig. 7B, top left panel). Antibodies in the

sera of animals immunized with antibody 1B1, on the other hand,

bound both nuclear and cytoplasmic antigens (Fig. 7B, top right

panel). In contrast, minimal binding was observed when serum

obtained from animals immunized with adjuvant was employed

(Fig. 7B, bottom left panel). Antibodies in the sera of antibody-

1B1-immunized animals demonstrated significantly enhanced

recognition of Ro60, La and the U1-RNP A protein in ELISA

assays (Fig. 7B, bottom right panel). Though Ro60 was

recognized by antibody 1B1 (which also bound SmD; Fig. 3B),

the reactivity towards the latter two proteins indicated the

presence of newer specificities in the sera of animals immunized

with this antibody.

Evidence of such a diversification of anti-self-responses was

also obtained upon immunization of C57BL/6 animals with

antibody 2C11 (IgMk). While such sera demonstrated significant

IgG autoreactive responses, no appreciable IgM responses were

observed. Antisera bound both cytoplasmic and nuclear antigens

in contrast to the cytoplasmic reactivity demonstrated by

antibody 2C11. Sera generated upon immunization with the

adjuvant did not contain significant autoreactive antibody

specificities. Further analysis revealed the preferential recogni-

tion of La and the U1-RNP A Protein in anti-2C11 sera. Though

the latter was an antigen bound by antibody 2C11 (which also

bound Ro52 in a dominant fashion, Fig. 3A), the former repre-

sented a new specificity (data not shown).

V region analysis

Figure 8 provides nucleotide sequence data for the IgM

antibodies and Fig. 9 for the IgG antibodies. Significantly,

antibodies demonstrated closest homologies to distinct germline

segments (and/or combinations thereof, as shown in Fig. 10A)

for both light and heavy chains, possibly a reflection of the

distinct antigenic specificities observed. For Antibodies 2H8 and

1B1, the heavy chain D regions were unidentifiable; Fig. 10B

shows ratios of the mutation frequencies observed in the CDR and

framework regions (FWR); a value in excess of unity was

obtained for the heavy chain of the IgM antibody 1B4, on account

of amino acid replacements observed in the CDR1 and CDR2

regions. For the heavy chain of antibody 2H8, a numerical value

could not be assigned on account of the divisor being zero. The

CDR of the IgM light chains were not preferentially mutated. Both

the heavy and light chains of the IgG Antibodies 2C3 and 1B1

contained an increased number of somatic mutations in the CDR

compared with FWR. While the heavy chain of antibody 1B3 was

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Figure 3. Reactivity of the IgM (A and C) and IgG (B and D) monoclonal antibodies towards recombinant RNP autoantigens (A and B) andphospholipids (C and D) by ELISA. LPC, lysophosphatidylcholine; LPA, lysophosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethano-lamine; PC, phosphatidylcholine; PA, phosphatidic acid; CL, cardiolipin. Bars represent arithmetic means of triplicate, plotted along with standarderrors.

Eur. J. Immunol. 2008. 38: 3561–3574 Clinical immunology 3565

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

similarly mutated, its light chain was assigned a value close to

unity, indicating an absence of preferential somatic mutations in

the CDR.

Discussion

In the context of the immune system, the processes of negative

selection in the thymus [18] as well as the killing of virally

infected cells [19] involve activation of the apoptotic cascade.

Normally, apoptotic cells are recognized by professional phago-

cytes and cleared very efficiently. The molecules and processes

involved in the specific recognition by the phagocyte of the

products of cell death have begun to be elucidated [20].

Phagocytosis of apoptotic cells is associated with the regulation

of inflammatory responses [21]. Lupus appears to be character-

ized by excessive apoptosis as well as defective clearance of

apoptotic debris, both of which would be expected to lead to the

accumulation of self-antigens and, possibly as a consequence, an

enhanced susceptibility to autoimmune disease. The specific cell-

surface recognition of ‘‘late-stage’’ apoptotic cells (as defined by

the presence of apoptosis-specific moieties along with demon-

strated retention of plasma membrane integrity) by antibodies

has been previously reported both by us [22, 23] and by

other investigators [16, 24]. In this study, the IgG and IgM

apoptotic cell-specific murine monoclonal antibodies generated

1000.0

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Figure 4. (A) Binding of antibodies in sera from SLE patients (dashedlines) and normal subjects (solid lines) to Ro60 by direct-binding ELISA.(B) Competitive ELISA demonstrating the ability of antibodies in serafrom SLE patients (dashed lines), but not from normal subjects (solidlines), to compete with antibody 2C3 for binding to Ro60. The squarerepresents absorbance in the absence of sera.

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Figure 5. Phagocytic uptake by peritoneal macrophages of apoptoticthymocytes in presence of (A) IgG and (B) IgM monoclonal antibodies.The IgG mediated enhanced uptake (Student’s t-test: �po0.05; ��po0.009versus isotype control) while the IgM diminished uptake (Student’s t-test:�po0.05; ��po0.008; ���po0.0009 versus isotype control) of apoptotic cells.The Phagocytic Index, calculated as described in the text, was plotted asfold change over relevant isotype control antibodies, indicated by thehorizontal line with open symbols. In both figures, the diamondrepresents the Phagocytic Index when healthy, non-apoptotic cells wereemployed in the absence of antibodies. Bars represent arithmetic meansof triplicate, plotted along with standard errors.

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demonstrated cross-reactivity across several RNP autoantigens.

Extensive cross-reactivity of anti-RNP responses has also

been previously described by other investigators [25]. Cross-

reactivity of autoimmune antibodies across different classes of

molecules has also been reported. For example, antibodies to

dsDNA have been shown to bind phospholipids and peptides

[26, 27]. Antibody 2C11 (IgMk) bound LPC, while antibody 2C3

(IgG2bk) appeared to be more broadly reactive in terms

of anti-lipid specificity. LPC is synthesized and secreted by

apoptotic cells by the action of calcium-independent phosholi-

pase-A2. Lipid extracts obtained from apoptotic thymocytes

contain LPC along with other oxidized phospholipids, and

immunization of mice with such cells generates antibodies that

specifically bind apoptotic cells through recognition of oxidation-

specific epitopes [28]. In certain contexts, LPC has anti-

inflammatory properties [29] and may contribute to the

quiescent milieu of the apoptotic cell-phagocyte environment.

LPC also acts as a chemo-attractant for phagocytes [30], probably

helping them locate dying cells in vivo. Infusion of LPC has

been demonstrated to interfere with the clearance of apoptotic

cells in gld mice [31] and mice deficient in the G2A ‘‘effector’’

exhibit late-onset autoimmunity [32]. Spontaneously arising

autoreactivity against the lipid could thus have far-reaching

consequences.

Sera from lupus-prone mice have been shown to inhibit the

uptake of apoptotic cells [10], a finding of significance, since

apoptotic debris is thought to provide the autoantigenic stimulus

in lupus. IgM antibodies against low-density lipoproteins on

apoptotic cells can inhibit phagocytosis of apoptotic cells [33], as

can an IgM antibody derived from animals immunized with dying

cells [34]. Such studies and others [23, 35, 36] question whether,

in all cases, the uptake of apoptotic cells is strictly PS-dependent

or whether other moieties on dying cells may also contribute.

Antibodies such as IgM 2C11, which binds LPC as well as apop-

totic blebs (also shown to act as chemo-attractants for phago-

cytes) and can also efficiently prevent the phagocytic uptake of

apoptotic cells, could be envisaged to be powerful disease

perpetuators in an environment rendered pro-inflammatory, due

to bacterial infection, for example. The IgG antibodies generated

in this study (2C3, 1B1 and 1B3), in contrast to IgM, enhanced

the internalization of apoptotic cells, possibly by Fc-gR-mediated

mechanisms. IgG anti-phospholipid antibodies that specifically

target apoptotic cells have been shown to increase uptake by

monocytes and induce the release of TNF-a [17]; on the other

hand, IgG antibodies to RNP Ro and La were shown to inhibit

uptake of apoptotic cardiocytes by healthy cardiocytes [37], cells

that do not express Fc receptors. SLE sera as well as small nuclear

RNP-autoantibody and DNA-autoantibody immune complexes

have been shown to stimulate the production of inflammatory

cytokines via the stimulation of Toll-like receptors [38, 39]. In the

present study, in no instances were antibodies found to influence

cytokine production (data not shown). It would be interesting to

attempt correlation between the epitopic specificity of auto-

antibodies directed towards RNP and disease progression and

severity.

Evidence exists implicating the idiotypic network in the

initiation and progression of autoimmune responses. Immuniza-

tion with an anti-dsDNA antibody bearing the 16/6 idiotype led

to the generation of an anti-dsDNA response in non-autoimmune-

prone mice [40]. Immunization with a human monoclonal anti-

body recognizing Ro60 and Ro52 resulted in novel autoantigens

being targeted. Anti-idiotypic antibodies also bound the surface

of healthy cells, unlike the immunizing antibody, which specifi-

cally bound apoptotic cells [23]. We have now addressed this

issue in a system that offers an increased level of stringency;

immunization of syngeneic mice with murine antibodies would

be expected to generate primarily an anti-idiotypic response,

unlike immunization of animals with human antibodies, which

necessitates extensive absorptions to rid the elicited antibodies of

isotypic and allotypic reactivity. Antibodies 1B1 (IgG2ak) and

00

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Figure 6. (A) Influence of antibody 2C11 or antibody 1B4 (employed asan isotype control) upon the down-modulatory effects of LPC on LPS-induced TNF-a secretion (Student’s t-test: �po0.001 antibody 2C11versus Antibody 1B4). Antibodies were used at a concentration of20 mg/well. Ab.: antibodies. (B) Effects of varying concentrations ofantibody 2C11 (filled circles) and an isotype control antibody (anti-TNP(IgMk); open circles) on the transmigration of THP-1 cells towardssupernatant derived from apoptotic cells (Student’s t-test: �po0.007;��po0.003 antibody 2C11 versus isotype control). The filled squarerepresents transmigration of THP-1 cells towards supernatant derivedfrom healthy cells. Bars represent arithmetic means of triplicate,plotted along with standard errors.

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2C11 (IgMk) were both capable of evoking anti-self-responses

and diversification in the repertoire. The ELISA data on select

recombinant proteins are only indicative in nature, and more

comprehensive antigenic analysis using anti-idiotypic sera would

obviously be more revealing. Nevertheless, the results provide

evidence that, in a syngeneic system, the idiotypic network can

mediate epitope spreading in anti-RNP responses. One way in

which this may occur can be postulated. Antibodies reactive

towards one component could conceivably generate ‘‘internal

image’’ anti-idiotypic antibodies that bind to other component

molecule(s) of a macromolecular complex to which the inciting

antigen belongs. While such a scenario is possible, all specificities

that arise in experiments such as these cannot be predicted a

priori, on the basis of knowledge of known interactions.

While the anti-1B1, anti-idiotypic specificity could conceivably be

predicted (based on the known associations of Ro60 with La and

of SmD with the U1-RNP complex), such forecasts for the anti-

2C11, anti-idiotypic specificity would be erroneous. Unexpected

specificities also arise upon immunization with anti-DNA anti-

bodies [40]. It is conceivable that unappreciated (and currently

unpredictable) levels of idiotypic connectivity exist amongst

autoimmune antibody responses. Anti-idiotype-mediated epitope

spreading is clearly not a general phenomenon, however, as

immunization with antibody 2C3 (IgG2bk) does not result in

IgGIgM

Adjuvant

1B1 Anti-IdB

A

0.0

0.2

0.4

0.6

0.8Ro52

Ro60

SmB

SmD

La

U1-RNP A

Ab

sorb

ance

(45

0 n

m)

Eve

nts

FL

1B1

Figure 7. (A) Assessment of IgM (left panel) and IgG (right panel) autoreactivity of sera isolated from NZB/W animals immunized with antibody 1B1(solid, thick profiles) or adjuvant (dashed profiles) by FACS analysis. Solid, thin profiles depict negative controls, staining obtained when just thesecond antibody was employed. FL: log fluorescence. (B) Top left: reactivity of antibody 1B1 upon permeabilized U-87 cells by immunofluorescence.Top right: reactivity of anti-1B1 sera. Bottom left: reactivity of adjuvant-induced sera. Bottom right: differential reactivity of anti-1B1 sera towards apanel of recombinant antigens by ELISA. Results are plotted after respective reactivity obtained upon immunization with adjuvant alone has beensubtracted, and so represent anti-idiotypic responses. Bars represent arithmetic means of triplicate, plotted along with standard errors.

Eur. J. Immunol. 2008. 38: 3561–3574Joy Das et al.3568

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Figure 8. Heavy and light chain variable region sequences for the IgM antibodies (A) 2C11, (B) 2H8 and (C) 1B4. FWR and CDR are indicated.Homologies to respective closest germline genes are indicated as dots. Amino acid mutations are indicated in lower case. Underlines indicate non-encoded amino acids.

Figure 9. Heavy and light chain variable region sequences for the IgG antibodies (A) 2C3, (B) 1B3 and (C) 1B1. FWR and CDR are indicated.Homologies to respective closest germline genes are indicated as dots. Amino acid mutations are indicated in lower case. Underlines indicate non-encoded amino acids.

Eur. J. Immunol. 2008. 38: 3561–3574 Clinical immunology 3569

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

anti-self-reactivity (data not shown). Further work, employing

antibodies of differing specificity and idiotypes, would be

required for a clearer appreciation of the rules that govern such

responses.

Distinct phagocytic outcomes can result, depending on the

nature of the targeted apoptotic cell-associated molecular

pattern, the nature of the phagocyte and the isotype of the

autoantibody, as this study and work by other investigators

suggest. Apoptotic cell-reactive antibodies can sometimes mani-

fest characteristics of an antigen-driven response, while still

demonstrating a variable degree of intermolecular cross-reactiv-

ity stretching across proteinic and lipidic molecular moieties.

While the basis of this cross-reactivity will form the focus of

future investigations, the current studies elaborate upon two

potential consequences of anti-lipid responses, the reversal of the

anti-inflammatory effects of LPC and the interruption of an

LPC-mediated phagocyte chemo-attractive signal, both of

which are clearly relevant in the context of systemic

autoimmunity. These studies also shed new light upon the

phenomenon of anti-idiotype-mediated determinant spreading;

the phenomenon is not dependent on antibody isotype or the

presence of somatic mutations in the CDR but is nonetheless not

universal. A more detailed analysis of the antibodies that bind

dying cells is clearly warranted in order to further understand the

consequences of the recognition of cell death in systemic auto-

immune disease.

Materials and methods

Human subjects

All procedures were reviewed and approved by Institutional

Ethics Committees and informed consent obtained.

SLE patients were on clinical followup at the Rheumatology

Clinic at the Department of Medicine at the All India Institute of

Medical Sciences, New Delhi. All subjects satisfied the revised

criteria set forth by the American College of Rheumatology for

the diagnosis of SLE and were evaluated and classified on the

basis of their SLE Disease Activity Index. Blood samples were

withdrawn from patients deemed to be in the midst of a disease

flare, as determined by clinical presentation. Control sera

samples were isolated from normal, healthy volunteers.

Generation of apoptotic cell-reactive murinemonoclonal antibodies

All procedures were reviewed and approved by Institutional

Ethics Committees.

All cultures were maintained in DMEM (Gibco BRL) supple-

mented with 10% FCS (Biological Industries). Cells were grown in

a humidified incubator at 371C in a 5% CO2 atmosphere. C57BL/

6lpr/lpr and NZB � NZW (F1) (hereafter referred to as NZB/W)

animals were maintained at the animal facility of the National

Institute of Immunology, New Delhi. Hybridomas were generated

by fusion of splenocytes (sourced from aging animals demon-

strating high serum anti-nuclear antibody titers) with SP2/0

myeloma cells in the presence of 50% v/v polyethylene glycol 1500

(Sigma). Hybrids were selected in hypoxanthene-, aminopterin-

and thymidine-supplemented medium for 7 days, and cells were

cultured for an additional week in medium supplemented with

hypoxanthene and thymidine. A total of 465 independent hybri-

domas were generated for this study. As an initial assessment of

reactivity, recognition of either healthy (non-permeable and non-

apoptotic) cells, as well as of permeabilized cells, was assessed by

flow cytometry; 32 hybridomas secreted antibodies reactive to the

surface of healthy cells, and 64 secreted antibodies that bound

permeabilized cells. Of the latter, 58 did not recognize apoptotic

cells (reflective of anti-self-reactivity towards nuclear or cyto-

plasmic moieties not necessarily involved in, or affected by, the

apoptotic cascade), while the six antibodies discussed in this paper

bound cells in the late stages of apoptosis.

Hybridoma cells secreting antibodies of interest were

subcloned at least three times by limiting dilution to

ensure monoclonality.

Induction of apoptosis

Human Jurkat T cells or murine SP2/O cells were exposed to

UV light (312 nm; 100 mJ/cm2), whereas murine thymocytes

J28-19JH2U.I.VH7183.101B1

J2ba9JH4DSP2.2J558.26.1161B3

J2bl1JH4DSP2.5VH7183.3b2C3

J423-48JH4DSP2.2J558.84.1901B4

J519-23JH3U.I.J558.67.1662H8

J1cp9JH1DFL16.1VH9.152C11

JLVLJHDHVHA

B

IgM

sIg

Gs

0

2

4

6 Heavy ChainLight Chain

IgMs IgGs2H8 *

CD

R/F

WR

Rep

lace

men

tM

uta

tio

n F

req

uen

cy

1B11B3 2C31B4 2C11

Figure 10. (A) Description of antibody germline segment usage (VH,DH and JH regions for heavy chains and VL, JL regions for light chains).U.I.: unidentifiable. (B) Ratios of CDR to FWR replacement mutationfrequencies in the six antibodies. � indicates a divisor of zero. See textfor details.

Eur. J. Immunol. 2008. 38: 3561–3574Joy Das et al.3570

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

were cultured in medium supplemented with 10 mM dexametha-

sone. In both instances, cells were then incubated for

different periods of time. At the time of analysis, apoptotic cells

retained the capacity to exclude the vital dye Trypan

Blue, indicating retention of plasma membrane integrity.

To establish the dependence of antibody binding upon

caspase activity, apoptosis was induced in the presence of

25 mM z-VAD-fmk, a pan-caspase inhibitor.

Antigenic reactivity

Flow cytometry and confocal analysis

All incubations were carried out in FACS buffer (PBS

containing 1% BSA and 0.2% sodium azide) for 1 h at 41C.

Healthy, apoptotic or permeabilized cells (obtained by

brief incubation in chilled methanol containing 0.001% Triton-

X-100) were incubated with antibodies and subsequently

washed to remove unbound antibody by brief centrifugations

at 300g. Fluorochrome-labeled secondary antibodies were

then added at the recommended dilution and another incubation

carried out. After further washes, cells were resuspended

in PBS and analyzed on a Becton Dickinson LSR flowcyto-

meter. To assess exposure of PS, antibody-stained cells

were first resuspended in Annexin-binding buffer (10 mM

Hepes, 140 mM NaCl and 2.5 mM CaCl2) followed by incubation

at room temperature for 20 min with an Annexin-V–fluorochrome

conjugate.

For confocal microscopy, apoptotic Jurkat T were fixed in

freshly prepared ice cold 2% paraformaldehyde in PBS for

10 min, followed by incubation in PBS supplemented with 1%

BSA for 15 min. Cells were then processed as described for flow

cytometry.

Western blot

Apoptotic blebs were prepared by differential centrifugation.

Briefly, apoptosis was induced in SP2/0 cells by UV light

as described under "Induction of apoptosis". Cells were

centrifuged at 400g for 5 min at 41C and the supernatant

re-centrifuged at 1000g for 10 min. The resulting supernatant

was further centrifuged at 17 000g for 30 min. The pellet

contained apoptotic blebs of 6–8 mm, as assessed by scanning

electron microscopy. Blebs (5mg protein) were incubated

with antibody 2C11 for 2 h at 371C and then subjected to

extensive washing to remove unbound antibody. SDS-PAGE

and Western blot were carried out, and the presence of

bleb-bound antibody was revealed by an anti-mouse-Ig-HRP-

secondary antibody conjugate. Additionally, blebs were isolated

from apoptotic 2C11 hybridoma cells and the presence of

endogenous, adhered antibody 2C11 was assessed by SDS-PAGE

and Western blot. In this case, blebs isolated from apoptotic

SP2/0 cells, which were not expected to contain adhered Ig,

served as negative controls.

ELISA

Recombinant autoantigens (Ro52, Ro60, SmB, SmD, La and

U1-RNP A Protein, obtained from Dr. Shu Man Fu, University of

Virginia, USA) were diluted in 0.1 M carbonate buffer, pH 9.2.

Fifty microliters of buffer containing 500 ng antigen

was dispensed in each well of a 96-well ELISA plate (Nunc).

Plates were incubated overnight at 41C. After wells were

washed with 10 mM PBS (pH 7.4) containing 0.05% Tween 20,

un-occupied binding sites were ‘‘blocked’’ by incubation with

200mL of 1% BSA prepared in PBS; this and all subsequent

incubations were at 371C for 2 h; 50 mL of primary antibodies

(murine monoclonal antibodies, SLE sera or normal

sera), appropriately diluted in PBS with Tween 20 containing

1% BSA, were dispensed into the wells and a further incubation

carried out. After further washes, 50 mL of diluted secondary

antibody was added to each well. Following another incubation,

wells were extensively washed. Enzymatic reactivity was

visualized with a tetramethyl benzidine, tetramethyl ammonium

borohydride and N,N-dimethyl acetamide containing substrate

solution. Absorbance was recorded at 450 nm. For competition

assays, diluted SLE sera or normal sera were pre-incubated in

antigen-adsorbed wells for 30 min followed by addition

of the monoclonal antibodies. Incubation was carried on for

another 90 min and reactivity was assessed following incubation

with a goat anti-mouse IgG-HRP conjugate (Jackson Immuno-

Research).

The following phospholipids were obtained from

Avanti Polar Lipids: LPC, lysophosphatidic acid (LPA), PS, phos-

phatidylethanolamine (PE), phosphatidylcholine (PC), phospha-

tidic acid (PA) and cardiolipin (CL). Phospholipids were

resuspended in 1:4 chloroform:methanol at a concentration of

50 mg/mL; 30 mL of this solution was dispensed into wells of

a 96-well ELISA plate. Evaporation of the solvent occurred during

incubation at 16 h at 41C. Un-occupied binding sites were

‘‘blocked’’ by incubation with PBS containing 5% low-fat milk

powder or 0.1% polyvinyl alcohol. Other procedures were as

mentioned for recombinant autoantigens.

Negative controls for all assays included antigen-blank

wells, and wells in which only antigen and second antibodies

were employed. Highest absorbances (which typically

ranged from 0.05 to 0.08) were subtracted.

Phagocytosis

Murine thymocytes were incubated with 10 nM CFSE diacetate

salt mixed isomer (Molecular Probes) for 20 min at 371C. Cells

were washed twice with RPMI-1640 and apoptosis was induced

by the addition of dexamethasone as described under ‘‘Induction

of apoptosis’’.

C57BL/6 mice were injected intraperitoneally with 1 mL of a

4% thioglycolate solution. Peritoneal exudates cells were

‘‘tapped’’ at 72 h. Cells were resuspended in DMEM and allowed

to adhere to tissue culture plates for 2 h. Non-adherent cells were

Eur. J. Immunol. 2008. 38: 3561–3574 Clinical immunology 3571

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

removed by repeated flushing. CFSE-labeled apoptotic thymo-

cytes were added to adhered phagocytes at a ratio of 1:4

(phagocytes:apoptotic cells) in the presence of varying concen-

trations of antibodies. An IgMk antibody monoclonal antibody

recognizing the Vi molecule of Salmonella typhi was employed as

the negative control for Antibodies 2C11, 2H8 and 1B4. Antibody

1D1 (an IgG3k monoclonal antibody generated in the laboratory,

which is reactive to cell-surface moieties upon healthy cells) was

employed as negative control for Antibodies 2C3, 1B3 and 1B1.

Cultures were incubated for 2 h at 371C and then transferred to

41C to stop further uptake. In certain instances, supernatants

were stored for subsequent cytokine analysis. Non-phagocytosed

thymocytes were removed by extensive ‘‘flushing’’. Cells were

stained with an anti-Mac1-phycoerythrin conjugate (PE; BD

Pharmingen) to label the macrophage population. FACS

analysis was then carried out. The Phagocytic Index was assessed

as the number of dually stained cells (macrophages (red) that

had ingested apoptotic cells (green)) over total number of

macrophages (red).

Inflammation

One of the apoptotic cell-specific antibodies (antibody 2C11

(IgMk)) was reactive towards LPC, a bioactive lipid that exhibits

both anti-inflammatory [29] and pro-inflammatory [41] proper-

ties. To delineate the anti-inflammatory effects of LPC in initial

experiments, thioglycolate-elicited peritoneal cells were cultured

in X-Vivo 10 serum-free media and allowed to adhere for 2–3 h at

371C; 100 ng/mL of S. typhi LPS (Sigma) was added to the cells,

along with varying concentrations of LPC or PC. An incubation

was then carried out for 4 h at 371C. LPC, but not PC, caused a

dose-dependent decrease in the secretion of TNF-a (data not

shown), estimated by ELISA (BD OptEIA). To assess the potential

of antibody 2C11 (as opposed to the apoptotic cell-specific but

LPC non-reactive antibody 1B4 (IgMk)) to revert the anti-

inflammatory effects of LPC, 20 mg of the respective antibodies

were added to cultures containing cells, LPS (100 ng/mL) and

LPC (50 mM), and TNF-a was estimated by ELISA.

Transmigration

Apoptotic cells are known to secrete LPC, which, in addition to its

other biological activities, also acts as a chemo-attractant for

phagocytic cells [30]. It is also believed that small membranous

vesicles released by apoptotic cells may have similar properties.

Since the antibody 2C11 bound both LPC and apoptotic

blebs, the ability of the antibody to inhibit the transmigration

of THP-1 cells towards supernatant obtained from apoptotic cell

cultures was assessed. An anti-TNP antibody (IgMk; BD

Pharmingen) was employed as the isotype control. Apoptosis

was induced in Jurkat cells as described under ‘‘Induction of

apoptosis’’. Supernatants were harvested by centrifugation at

1200g for 10 min; 200mL of apoptotic cell-culture supernatant

(either non-supplemented or supplemented with varying concen-

tration of antibody 2C11 or the isotype control antibody) was

added to the lower chamber of transmigration wells. THP-1 cells

(50 mL of a suspension of 2�106 cells/mL) were added to

transwell inserts (8 mm pore size; BD Falcon) and an incubation

carried out for 90 min under standard culture conditions.

Transmigration was assessed by counting the cells harvested

from the lower chamber.

Anti-idiotypic responses

Antibody 2C11 (IgMk) was purified by affinity chromatography

on UltraLink Immobilized Mannan Binding Protein (Pierce).

antibody 1B1 (IgG2ak) and antibody 2C3 (IgG2bk) were purified

using Protein-G Sepharose affinity chromatography (GE Health-

care). Six-wk-old animals (C57BL/6 for Antibodies 2C11, and

NZB/W for Antibodies 1B1 and 2C3) received subcutaneous

injections of 50 mg antibody emulsified in Incomplete Freund’s

Adjuvant; each animal received 100mL at two sites. Two booster

injections were administered at weekly intervals and blood

samples were collected 1 wk after each booster. Anti-self-

reactivity in the sera was assessed by FACS analysis on

permeabilized and non-permeabilized Jurkat cells, by indirect

immunofluorescence assays on HeLa cells and by ELISA upon a

panel of recombinant autoantigens.

Antibody variable region genes

RNA was isolated from extensively subcloned hybridoma cultures

by the TRIzol (Invitrogen) method. cDNA was prepared using

oligo dT primers and avian myeloblastosis virus reverse tran-

scriptase (Promega). RNA and the oligo dT primer (Promega)

were incubated at 701C for 10 min followed by instantaneous

cooling on ice. Additional pre-mixed components were added and

the mixture incubated on a thermal cycler (Perkin Elmer) for

60 min at 481C. Reverse transcriptase was inactivated by heating

to 701C for 5 min. The cDNA preparation was treated with RNase

H (Promega) at 371C for 30 min to degrade residual RNA. A

mouse Ig kit (Novagen) containing primers for the murine light

and heavy chain genes was employed. Forward and reverse

primers (5 pM) were added in a total reaction volume

of 25 mL. PCR amplification was then carried out (30 cycles;

5 min hotstart, 45 s denaturation at 941C, 45 s annealing at 601C).

Extension was carried out for 45 s at 721C; a final extension for

7 min was carried out at 721C. After electrophoresis on 1%

agarose, the PCR product was purified using the Qiagen gel

extraction kit and ligated to the pGEMT vector (Promega). The

DH5a strain of Escherichia coli was transformed with the ligated

plasmid using standard protocols. The transformed cells were

selected on LB agar supplemented with ampicillin (100 mg/mL),

X-gal and iso-propyl thio-galactopyranose. Plasmids were isolated

and cloned antibody gene inserts were sequenced by using M13

(forward and reverse) primers, corresponding to sequences

Eur. J. Immunol. 2008. 38: 3561–3574Joy Das et al.3572

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

adjacent to the multiple cloning site of the pGEMT vector.

Automated sequencing was carried out at the Centralized

Sequencing Facility of the National Institute of Immunology.

Analysis of antibody variable region gene sequences

was carried out using the NCBI and IMGT germline databases.

Sequences were submitted to GenBank (Accession Numbers

EF063577–EF063588; http://www.ncbi.nlm.nih.gov/Genbank).

Mutation frequencies (cumulative replacement mutations per

unit amino acid length) were calculated for the CDR and FRW

regions; in order to obtain a conservative estimate, amino acids

not unequivocally assignable to designated gene segments were

not included in the analysis.

Acknowledgements: This study was supported by core and

extramural grants from the Department of Biotechnology,

Government of India to R. P.

Conflict of interest: The authors have declared no financial or

commercial conflict of interest.

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Abbreviations: LPC: lysophosphatidylcholine � PC: phosphatidylcholine

� PE: phosphatidylethanolamine � PS: phosphatidylserine � RNP:

ribonucleoprotein � SLE: systemic lupus erythematosus

Full correspondence: Dr. Rahul Pal, Immunoendocrinology Laboratory,

National Institute of Immunology, Aruna Asaf Ali Marg, JNU Complex,

New Delhi, India

Fax: 191-11-26742125

e-mail: rahul@nii.res.in

Received: 18/6/2008

Revised: 27/8/2008

Accepted: 29/9/2008

Eur. J. Immunol. 2008. 38: 3561–3574Joy Das et al.3574

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu