The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 5250-5256
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moll, H. P.
Right arrow Articles by Brostjan, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moll, H. P.
Right arrow Articles by Brostjan, C.

Neutralizing Type I IFN Antibodies Trigger an IFN-Like Response in Endothelial Cells1

Herwig Peter Moll2, Harald Freudenthaler2, Anna Zommer, Elisabeth Buchberger and Christine Brostjan3

Department of Surgery Research Laboratories, Medical University of Vienna, Vienna General Hospital, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neutralizing Abs to type I IFNs are of therapeutic significance, i.e., are currently evaluated for the treatment of autoimmune diseases with pathogenic IFN-{alpha} production such as for systemic lupus erythematosus. Unexpectedly, we observed that several neutralizing Abs reportedly known to counteract IFN-{alpha} or IFN-β activity triggered an "IFN-like" response in quiescent primary human endothelial cells leading to activation of the transcription factor IFN-stimulated gene factor 3 and the expression of IFN-responsive genes. Furthermore, these Abs were found to enhance rather than inhibit type I IFN signals, and the effect was also detectable for distinct other cell types such as PBMCs. The stimulatory capacity of anti-IFN-{alpha}/β Abs was mediated by the constitutive autocrine production of "subthreshold" IFN levels, involved the type I IFNR and was dependent on the Fc Ab domain, as Fab or F(ab')2 fragments potently inhibited IFN activity. We thus propose that a combined effect of IFN recognition by the Ab paratope and the concomitant engagement of the Fc domain may trigger an IFN signal via the respective type I IFNR, which accounts for the observed IFN-like response to the neutralizing Abs. With respect to clinical applications, the finding may be of importance for the design of recombinant Abs vs Fab or F(ab')2 fragments to efficiently counteract IFN activity without undesirable activating effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The inducible IFN response and the associated antivirus, antitumor, and immunomodulatory activities are well-characterized hallmarks of the defense system. These activities are primarily mediated by the rapid activation of the transcription factor IFN-stimulated gene factor (ISGF)3,4 which binds to promoter IFN-stimulated response elements (ISREs) and induces expression of IFN response genes such as IFIT-1 (IFN-induced protein with tetratricopeptide repeats 1) or IFN-stimulated gene (ISG)15. In contrast, the low level, constitutive expression of type I IFNs (IFN-{alpha} or IFN-β) has now been recognized to serve distinct functions in cellular signaling and activation (1): in the absence of any known stimulus, a low basal expression level of type I IFNs is maintained, which results in a weak signaling event and intracellular tyrosine phosphorylation of the type I IFN receptor {alpha}-chain (IFNAR-1). The signal is considered to be "subthreshold," namely does not elicit the signaling cascade that leads to transcriptional activation of IFN response genes. However, IFNAR-1 is maintained in a "ready state" thereby promoting rapid cellular activation upon stimulation with virus, IFNs or other STAT signaling cytokines. In this context, IFNAR-1 was shown to engage in cross-talk with other receptors such as the type II IFN-{gamma} or the IL-6R, thus constituting a common "docking site" for STAT dimerization and an efficient enhancer of cellular activation (2, 3). Constitutive, low level IFN-{alpha}/β expression has been reported for mouse embryonic fibroblasts, splenocytes, macrophages, and bone marrow cells (2, 3), and was further described to promote activation of CD8+ T cells upon TCR engagement (4) and to regulate dendritic cell differentiation (5, 6).

Considering the potency of IFN-{alpha}/β in the immune response, the application of recombinant IFN (rIFN) has proven an evident and valuable therapeutic tool in the treatment of, e.g., viral infections or cancer. In contrast, type I IFNs, in particular IFN-{alpha}, have been found to play a crucial role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus, type-1 diabetes, or autoimmune thyroid disease (7, 8). In this context, the development of a neutralizing Ab directed against multiple IFN-{alpha} subtypes has been promoted (9), and in April 2006 the first clinical trial has been launched applying a humanized IFN-{alpha} blocking mAb to systemic lupus erythematosus patients. IFN-neutralizing activities are expected to target immune effector cells such as leukocytes, but their impact further extends to other cell types such as the vessel-lining endothelial cells (ECs). In this regard, we have observed and characterized an unexpected "IFN-like" response in ECs upon exposure to neutralizing Abs directed against type I IFNs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

Primary ECs were isolated from human foreskin samples by dispase digest, purified via anti-CD31 Ab-coupled Dynabeads (Invitrogen Life Technologies) and cultured in fibronectin-containing Microvascular Endothelial Cell Growth Medium EGM2-MV (Cambrex) without vascular endothelial growth factor supplementation. Purified EC cultures showed ≥98% purity and viability. For separation of lymphatic and blood vessel ECs, anti-podoplanin Ab-coupled Dynabeads were applied. All isolates were characterized by flow cytometry for EC characteristics, i.e., CD31 expression, CD34 as marker of microvessels and for E-selectin induction following stimulation with 100 ng/ml TNF-{alpha} for 4 h. Lymphatic ECs were detected by podoplanin expression. To keep the passage number low, experiments were generally repeated with different EC isolates, i.e., represent biological replicates with variable inducibility. Representative results of two to four comparable experiments are shown.

HUVECs were obtained from Cambrex and grown in fibronectin-containing EGM2 medium without vascular endothelial growth factor supplementation (Cambrex). 293T cells are derived from human embryonic kidney cells into which the temperature sensitive gene for simian virus SV40 tumor Ag was inserted. HT-29 was isolated from a human colorectal adenocarcinoma. Both cell lines (293T, HT-29) were supplied by American Type Culture Collection and cultured in DMEM with 10% FCS. PBMCs were isolated from 100 ml EDTA-treated whole blood of a healthy volunteer by standardized density gradient centrifugation using Ficoll-Paque (GE Healthcare) and were supplied with RPMI 1640 medium containing 10% FCS.

Treatment with neutralizing Abs and rIFN

Two days before stimulation, ECs were seeded in growth medium at 7 x 105 cells per 30-mm dish (293T and HT-29 cells at 2 x 106 cells) to yield a confluent cell layer within 24 h. Culture medium was then exchanged to EGM2-MV containing 5% FCS but no additional growth factor supplements and cells were allowed to adopt a quiescent state over the next 24 h. Abs were added on day 3 at various concentrations and for the time periods indicated. A total of 7 x 105 PBMCs in 500 µl of RPMI 1640 medium were stimulated immediately after isolation without prior culture period.

The rIFN and blocking mAbs targeting either IFN-{alpha} (clones 2 and 13, MMHA-2 and MMHA-13, respectively) or IFN-β (clones 3 and 12, MMHB-3 and MMHB-12, respectively) as well as the IFNAR chain 2 (MMHAR-2) were all obtained from PBL Biomedical Laboratories. ELISA kits for detection of human IFN-{alpha} or IFN-β were also manufactured by PBL Biomedical Laboratories and assays were conducted essentially as described (10). Neutralizing Abs to IFN-{gamma} (NIB42) or the IFN-{gamma} receptor (IFNGR) {alpha}-chain (GIR-208) were supplied by BD Biosciences. For mouse IgG1 isotype control, the MOPC-21 clone was applied (Sigma-Aldrich). TNF-{alpha} was provided by H. R. Alexander (National Cancer Institute, Bethesda, MD), whereas LPS was obtained from Sigma-Aldrich.

Real-time RT-PCR

Total RNA was isolated from cell cultures with RNeasy Mini kit (Qiagen), 500 ng of RNA were reverse transcribed with oligo(dT) primers using the Superscript III First Strand Synthesis System (Invitrogen Life Technologies) and the generated cDNA was diluted 1/25 before PCR analysis. Real-time PCR was performed with SYBR Green PCR Core Reagents (Applied Biosystems) and the following primer sets: IFIT-1 (900 nM forward) 5'-GCA GAA CGG CTG CCT AAT TT-3', (900 nM reverse) 5'-TCA GGC ATT TCA TCG TCA TC-3'; ISG15 (300 nM forward) 5'-GAG AGG CAG CGA ACT CAT CT-3', (300 nM reverse) 5'-AGC TCT GAC ACC GAC ATG G-3'; IFN-β (50 nM forward) 5'-AGC ACT GGC TGG AAT GAG AC-3', (300 nM reverse) 5'-TCC TTG GCC TTC AGG TAA TG-3'; IFN-{alpha}1 (300 nM forward) 5'-GCC TCG CCC TTT GCT TTA CT-3', (300 nM reverse) 5'-CTG TGG GTC TCA GGG AGA TCA-3' (PrimerBank ID no. 13128950a1); IFN-{alpha}2 (300 nM forward) 5'-GCT TGG GAT GAG ACC CTC CTA-3', (300 nM reverse) 5'-CCC ACC CCC TGT ATC ACA C-3' (PrimerBank ID no. 11067751a1); BCoR (300 nM forward) 5'-AGA CGA CAT GCT CTC AGC AA-3', (50 nM reverse) 5'-GAT CCT ATG GGC CGT GCT 3'; housekeeping genes β2-microglobulin (50 nM forward) 5'-GAT GAG TAT GCC TGC CGT GTG-3', (50 nM reverse) 5'-CAA TCC AAA TGC GGC ATC T-3'; and β-actin (900 nM forward) 5'-CTG GAA CGG TGA AGG TGA CA-3', (300 nM reverse) 5'-AAG GGA CTT CCT GTA ACA ATG CA-3'. With the exception of IFNs, which do not contain introns, all primer sets span at least one exon/intron gene boundary. Primer sets for IFN-{alpha}1 and IFN-{alpha}2 were retrieved from the PrimerBank (11). Each sample was assayed in triplicate with the GeneAmp 5700 Sequence Detection System (Applied Biosystems) for 45 cycles of 15 s at 95°C followed by 1 min at 60°C and a final dissociation protocol to screen for false amplification products. The mRNA levels for IFIT-1, ISG15, BCoR, or IFNs were deduced from the on-plate dilution series of a standard cDNA and were normalized to housekeeping gene values as previously described (12). Real-time PCR data are given as mean and SD of triplicate samples. The value obtained for the untreated control sample was generally set to 1 and changes in mRNA expression upon stimulation are given in relation to the untreated control.

Immunoblotting

Endothelial whole cell extracts were generated essentially as described (13, 14), i.e., in lysis buffer containing 1% Nonidet P-40, 0.5% deoxycholic acid, and the Complete MiniProtease Inhibitor Cocktail (Roche). The 30 µg of total protein were separated on PAGE minigels and subjected to wet blotting. Immunodetection was performed with polyclonal rabbit antiserum against IFIT-1, a gift by G. Sen (Cleveland Clinic, Cleveland, OH) at a 1/2000 dilution or ISG15 (Rockland Immunochemicals) at a 1/200 dilution.

EC transfection with small interfering siRNA

Three distinct Stealth siRNA duplex oligonucleotides for IFN-β gene silencing (HSS10523-2, HSS10523-3, HSS10523-4) as well as the respective negative control siRNAs with matched (guanine-cytosine) GC content were obtained from Invitrogen Life Technologies. A total of 2 x 106 proliferating ECs were harvested in 400 µl of RPMI 1640 with 10% FCS and siRNA was added. Cells were then subjected to electroporation at 200 V, 960 µF essentially as described (15). Stimulation of ECs with anti-IFN mAbs or rIFN was performed 24–48 h posttransfection. A mix of the three IFN-β siRNAs (1 µM each) proved to be more effective than single application of the IFN-β siRNA variants (data not shown).

ISGF3 reporter gene assay

Electroporation of 2 x 106 ECs at 200 V, 960 µF was performed with a total of 30 µg of DNA. A combination of 29 µg of pISRE-Luc plasmid with five ISRE sites directing expression of the luciferase reporter gene (Stratagene) and 1 µg of the constitutive β-galactosidase expression plasmid pCMVβ for normalization (Clontech Laboratories) was applied. Cells were then seeded in 30-mm wells at 1 x 106 to yield a confluent cell layer within 24 h. Stimulation with anti-IFN-{alpha} mAb clone no. 2 (6 µg/ml) or rIFN-{alpha} (100 pg/ml) was conducted for 4 and 24 h. Cells were then harvested in 40 µl of lysis buffer and samples (10 µl) were assayed in triplicate for luciferase as well as β-galactosidase activity applying the Tropix Dual-Light System according to the manufacturer’s instructions (Applied Biosystems) for chemiluminescent detection with a Wallac Victor multilabel counter (PerkinElmer Life Sciences). Luciferase activity as measured in relative light units was normalized to the corresponding β-galactosidase value.

Generation of Fab and F(ab')2 fragments

The anti-IFN-{alpha} mAb clone no. 2 or mouse IgG1 isotype control Ab were subjected to ficin digest at 37°C in the presence of 10 mM cysteine for Fab vs 1 mM cysteine for F(ab')2 fragment generation according to the instructions of the ImmunoPure IgG1 Fab and F(ab')2 Preparation kit (Pierce). For control, an Ab fraction was treated comparably in ImmunoPure Digestion buffer but without addition of ficin protease. The resulting Ab fragments were evaluated under reducing as well as nonreducing conditions by Western blotting with ImmunoPure peroxidase-conjugated goat anti-mouse IgG (H+L chain) antiserum (Pierce) and digest efficiency was found to be ≥90%. Control human Fab/{kappa} fragments were obtained from Bethyl Laboratories.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Primary ECs as isolated from human dermal microvessels (HDMECs) were subjected to standard in vitro culture. When cells were exposed to increasing doses of neutralizing Abs directed against IFN-{alpha} or IFN-β (in the absence of any exogenously added type I IFN) an "IFN-like" response was observed (Fig. 1): the dose-dependent induction of IFN-responsive genes such as IFIT-1 and ISG15 was detected at the mRNA level by real-time RT-PCR as well as at the protein level by immunoblotting. The effect was verified for four different, commercially available blocking mAbs targeting either IFN-{alpha} (clone nos. 2 and 13) or IFN-β (clone nos. 3 and 12) as illustrated in Fig. 1 (also see Fig. 4). Furthermore, the stimulatory capacity of anti-IFN-{alpha} and anti-IFN-β mAbs was additive (Fig. 2A) and IFIT-1 mRNA induction was observed at all time points (2, 4, 6, and 8 h) investigated (data not shown). All mAbs tested were of the mouse IgG1 isotype with {kappa} light chain. An appropriate isotype control did not induce IFIT-1 or ISG15 expression in HDMECs.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 1. Dose-dependent induction of IFN-responsive genes by neutralizing Abs to type I IFN. EC cultures were treated for 4 h with 0.8, 3, or 12 µg/ml anti-IFN-{alpha} mAb clone no. 2 (n = 7 comparable experiments) (A), clone no. 13 (n = 2 comparable experiments) (B), or anti-IFN-β mAb clone no. 3 (n = 3 comparable experiments) (C). The nonspecific mouse IgG1 isotype control was applied at 12 µg/ml. Real-time RT-PCR analysis of endothelial RNA was performed to evaluate mRNA levels of IFIT-1 and ISG15. Each sample was assayed in triplicate, and comparable experiments were performed. IFIT-1 induction by Ab treatment was statistically significant (p ≤ 0.03) for all mAbs and concentrations. Comparably, p ≤ 0.01 was recorded for ISG15 induction with the exception of anti-IFN-{alpha} mAb clone no. 13 at the lowest concentration (p was not significant). D, Protein expression of IFIT-1 and ISG15 was investigated by immunoblotting of whole cell extracts following 5 h of stimulation with anti-IFN-{alpha} mAb clone no. 2 at 12 µg/ml (n = 2 comparable experiments). An asterisk indictes nonspecific, cross-reactive protein.

 

Figure 2
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 2. Characterization of the mechanisms involved in the induction of IFN response genes by neutralizing Abs to type I IFN. A, HDMECs were challenged for 4 h by single or combined treatment with 3 µg/ml anti-IFN-{alpha} mAb clone no. 2 and anti-IFN-β mAb clone no. 3 (or mouse IgG1 isotype control) (n = 3 comparable experiments). Endothelial RNA and corresponding cDNA were then analyzed for IFIT-1 mRNA expression by real-time PCR, which demonstrated an additive effect of anti-IFN-{alpha} and anti-IFN-β Abs. B, IFIT-1 induction by 3 µg/ml anti-IFN-{alpha} mAb clone no. 2 was blocked by the addition of anti-IFNAR Ab at 1 µg/ml vs mouse IgG2a isotype control (n = 3 comparable experiments). C, Activation of the IFN responsive transcription factor ISGF3 was evaluated by reporter gene assay measuring luciferase activity in relative light units (RLU) after EC stimulation for 4 or 24 h with 6 µg/ml anti-IFN-{alpha} mAb clone no. 2 or 100 pg/ml rIFN-{alpha} for comparison (n = 2 comparable experiments). D, Endothelial cultures after 0, 4, or 24 h of stimulation with 6 µg/ml anti-IFN-{alpha} mAb clone no. 2 were investigated by phase contrast microscopy to document that no apparent changes in EC morphology were triggered by Ab treatment.

 
A first indication on the potential mechanism underlying the extraordinary "intrinsic" ability of the type I IFN Abs to induce IFN-responsive genes came from the observation that IFIT-1 induction by anti-IFN-{alpha} or anti-IFN-β mAbs was abolished in the presence of IFNAR blocking Abs (Fig. 2B). Thus, the type I IFNR seemed to be involved even in the absence of exogenously added IFN. Furthermore, Ab treatment resulted in the activation of the ISGF3 transcription factor as monitored by reporter gene assay (Fig. 2C), indicating an IFN-like signal resulting in the transcriptional regulation of IFIT-1 and ISG15. Exposure of ECs to the IFN-neutralizing Abs was well tolerated and did not result in any apparent changes in morphology (Fig. 2D) or signs of apoptosis (data not shown) over prolonged time periods. Concomitant proinflammatory activation of ECs by LPS or TNF-{alpha} led to a partial reduction but could not prevent the endothelial IFN-like response to the Abs (Fig. 3A).


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 3. IFN-like response to neutralizing Abs by different EC variants or other cell types. A, HDMECs stimulated with proinflammatory mediators such as LPS at 1 µg/ml or TNF-{alpha} at 100 ng/ml were concomitantly treated with 12 µg/ml anti-IFN-{alpha} mAb clone no. 2 for 4 h (n = 2 comparable experiments). B, EC populations isolated from blood (BECs) or lymphatic (LECs) skin vessels or HUVECs were exposed to 12 µg/ml anti-IFN-{alpha} mAb clone no. 2 or mouse IgG1 isotype control for 4 h (n = 2 comparable experiments). C, Comparably, freshly isolated PBMCs or in vitro cultures of 293T and HT-29 cells were exposed to 12 µg/ml anti-IFN-{alpha} mAb clone no. 2 for 4 h (n = 2 comparable experiments). Total RNA and corresponding cDNA were analyzed for IFIT-1 mRNA expression by real-time PCR.

 
The tested cell isolates mostly consisted of a mixture of vascular and lymphatic ECs originating from human skin microvessels. When analyzed in separate cultures, comparable responses were elicited in both cell populations (Fig. 3B). In addition, endothelial cultures derived from larger vessels (HUVECs) were highly responsive to the type I IFN Abs. When investigating other human cell types, we found that the phenomenon was not restricted to ECs but could also be observed in freshly isolated PBMCs when treated with the respective IFN blocking mAbs (Fig. 3C). In contrast, the effect was essentially absent in Ab-treated 293T or HT-29 cell cultures representing a human embryonic kidney and colon carcinoma cell line, respectively.

Because all the Abs tested on HDMECs were established neutralizing monoclonals, we proceeded to test their blocking abilities in EC combination treatment with rIFN and mAb (Fig. 4). Four hours of incubation with rIFN-β (10 pg/ml) induced IFIT-1 mRNA levels by ~40-fold. Interestingly, addition of blocking mAbs (at 12 µg/ml) targeting IFN-β resulted in a further increase of IFIT-1 expression by 2- to 10-fold depending on the Ab applied (clone nos. 12 and 3, respectively). A similar phenomenon was observed when combining rIFN-{alpha} with anti-IFN-{alpha} blocking mAbs. In contrast, a neutralizing Ab directed against the type I IFNR, IFNAR, completely abrogated the endothelial response to rIFN-{alpha}. Furthermore, when IFIT-1 expression was triggered by type II rIFN (10,000 U/ml, rIFN-{gamma}), the induction was efficiently blocked by the addition of neutralizing Ab targeting either IFN-{gamma} or the corresponding type II receptor.


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 4. Enhanced response to rIFN-{alpha} or rIFN-β in the presence of neutralizing Abs to type I IFN. A, HDMECs were stimulated with rIFN-β (10 pg/ml) for 4 h in the absence or presence of anti-IFN-β mAb clone no. 3 or clone no. 12, or mouse IgG1 isotype control at 12 µg/ml each (n = 3 comparable experiments). B, EC treatment for 6 h with rIFN-{alpha} (10 pg/ml) was combined with 12 µg/ml mouse IgG1 isotype control, anti-IFN-{alpha} mAb clone no. 2 or anti-IFNAR Ab (n = 2 comparable experiments). C, Comparably, ECs were exposed for 6 h to 10,000 U/ml rIFN-{gamma} and 12 µg/ml mouse IgG1 isotype control, anti-IFN-{gamma}, or anti-IFNGR Ab (n = 3 comparable experiments). Endothelial RNA was analyzed for IFIT-1 mRNA expression by real-time RT-PCR.

 
As the ability of the type I IFN Abs to induce IFN-responsive genes was not dependent on but could be enhanced by exogenously added IFN, we hypothesized that ECs might constitutively express low levels of autocrine type I IFN contributing to the effects observed. To test our hypothesis, silencing of IFN-β gene expression was achieved by transiently transfecting HDMEC cultures with double-stranded siRNAs. When HDMECs were challenged with IFN-β siRNA or nonspecific (control) siRNA, no induction of IFIT-1 was observed, i.e., there was no endothelial response to the uptake of chemically modified dsRNA oligonucleotides (Fig. 5A). At 24 h posttransfection, ECs were exposed to anti-IFN-{alpha} or anti-IFN-β mAbs at 12 µg/ml. Induction of IFIT-1 mRNA was markedly inhibited by IFN-β gene silencing as opposed to control siRNA treatment (Fig. 5A): IFIT-1 expression levels in response to type I IFN Abs were reduced to ~40%. The IFN silencing efficiency of these samples was evaluated by real-time RT-PCR analysis and equaled the effect seen for IFIT-1. However, not only IFN-β mRNA levels but also IFN-{alpha}1 and IFN-{alpha}2 transcripts were decreased by ~60% in the presence of IFN-β siRNA (Fig. 5D). In contrast, the detectable mRNA expression of nonrelated EC genes, such as the transcriptional regulator BCoR or the housekeeping gene β-actin, was not affected by IFN-β vs control siRNA (Fig. 5E). To reverse the effect of IFN-β gene silencing, ECs were pretreated with subthreshold concentrations of IFN-β at 1 pg/ml (corresponding to 0.1 U/ml) for 2 h and were then challenged with anti-IFN-β blocking mAb (Fig. 5B). As expected, rIFN-β at 1 pg/ml was below the signal threshold, which was insufficient per se to induce IFIT-1 expression. However, the low-dose pretreatment restored EC responsiveness to anti-IFN-β-neutralizing mAb to 84%. Comparably, EC activation by high-dose (100 pg/ml) IFN-{alpha} was inhibited to 38% due to IFN-β gene silencing, whereas high-dose IFN-β could partially overcome the block and achieve expression levels of 76% (Fig. 5C).


Figure 5
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 5. IFN-β gene silencing inhibits the IFN-like response of ECs to IFN-neutralizing mAbs. A, Following EC transfection with IFN-β siRNA or nonspecific control siRNA, cells were challenged with 12 µg/ml anti-IFN-{alpha} mAb clone no. 2 or anti-IFN-β mAb clone no. 3 for 2 h (n = 3 comparable experiments). B, To reverse the effect of IFN-β gene silencing, EC cultures were pretreated with subthreshold concentrations of rIFN-β (1 pg/ml) for 2 h with or without subsequent addition of 12 µg/ml IFN-β mAb clone no. 3 for another 2 h (n = 2). C, Treatment of siRNA transfected ECs with high-dose (100 pg/ml) rIFN-{alpha} or rIFN-β for 4 h (n = 4 comparable experiments) was followed by RNA isolation and real-time RT-PCR detection of IFIT-1 expression. D, Silencing efficiencies for IFN-β as well as IFN-{alpha}1 and IFN-{alpha}2 were determined by real-time RT-PCR and are shown for EC samples stimulated for 2 h with 12 µg/ml anti-IFN-β mAb clone no. 3 (n = 3). E, Comparably, the effect of IFN-β siRNA vs nonspecific control siRNA was tested on transcript levels of nonrelated EC genes such as BCoR and β-actin for the same samples (n = 3 comparable experiments).

 
Having established that endogenously expressed type I IFN mediates the unexpected EC responsiveness to IFN-neutralizing mAbs, we then questioned whether Ab binding to Fc{gamma}Rs on the cell surface might contribute to the effects observed. Despite the fact that all our in vitro experiments were conducted in the presence of 5% FCS in culture medium thus supplying an excess of bovine IgG, binding of the mouse monoclonal anti-IFN Abs to human endothelial Fc{gamma}Rs could not be excluded. We therefore added increasing concentrations of mouse IgG1 isotype Ab to our reactions (Fig. 6B). A dose-dependent decline in EC responsiveness, i.e., in IFIT-1 mRNA induction by IFN-neutralizing mAbs, was observed. This response prompted us to further investigate the requirement for the Fc domain, thus we generated Fab as well as F(ab')2 fragments from anti-IFN-{alpha} blocking mAb clone no. 2 by ficin digest. When comparing the Fab and F(ab')2 fragments to the intact Ab (subjected to a mock treatment without addition of ficin protease for control) the intact molecule retained its dose-dependent stimulatory capacity, whereas the corresponding Fab or F(ab')2 fragments could not induce IFIT-1 mRNA expression in HDMECs (Fig. 6A). However, the generated fragments exhibited strong neutralizing capacity for rIFN-{alpha}, which was not observed when Fab or F(ab')2 fragments of the control mouse IgG1 isotype Ab were applied (Fig. 6C). IFIT-1 mRNA induction in response to EC treatment with 10 pg/ml rIFN-{alpha} was entirely abolished by 1 µg/ml Fab or F(ab')2 fragments from anti-IFN-{alpha} blocking mAb clone no. 2, which documents the actual IFN-neutralizing potency attributed to the original Ab by the manufacturer. In comparison, the fragments were less potent in competing, or inhibiting, the activity of the intact Ig molecule. Increasing the amount of Fab fragment while maintaining the concentration of intact anti-IFN-{alpha} mAb clone no. 2 at 1 µg/ml led to a 64% drop in IFIT-1 transcript levels at a ratio of 2:1. In contrast, a comparable amount of unrelated control Fab fragment led to a nonspecific quenching of IFIT-1 induction in the range of 25% (Fig. 6B).


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 6. Ab domains involved in IFIT-1 induction by IFN-neutralizing Abs. A, HDMECs were exposed for 4 h to 1, 3, or 6 µg/ml of intact (control-treated) anti-IFN-{alpha} mAb clone no. 2 or a generated Fab or F(ab')2 fragment of clone no. 2. Incubation for 4 h was followed by real-time RT-PCR analysis of endothelial RNA for IFIT-1 mRNA expression (n = 4 comparable experiments). In comparison to the full-length Ab, the fragments were incapable of eliciting IFIT-1 mRNA expression. p ≤ 0.029 for all concentrations of intact mAb vs fragments applied. B, The full-length anti-IFN-{alpha} mAb clone no. 2 (1 µg/ml) was mixed at a ratio of 1:1 or 1:2 with the corresponding generated Fab fragment, a nonspecific pool of unrelated Fab fragments or with an intact mouse IgG1 isotype control (n = 4 comparable experiments). C, HDMEC stimulation with rIFN-{alpha} at 10 pg/ml for 4 h was challenged with concomitant administration of 1 µg/ml Fab or F(ab')2 fragment generated from anti-IFN-{alpha} mAb clone no. 2 or from a mouse IgG1 isotype Ab (n = 3 comparable experiments).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the study presented, we have investigated the unexpected induction of IFN-regulated genes IFIT-1 and ISG15 in primary ECs that were exposed to Abs known to neutralize type I IFN. Despite the absence of exogenously added IFN, an "IFN-like" signal was observed involving the type I IFNR and leading to the activation of the transcription factor ISGF3. The potency of eliciting this response varied among the Abs applied, apparently relating to differences in binding affinities rather than the specificity for IFN-{alpha} or IFN-β. Depending on the clone, Ab concentrations in the range from 1 to 10 µg/ml were sufficient to induce IFIT-1 mRNA levels comparably achieved by EC stimulation with 10 pg/ml rIFN-β (40-fold). It is of interest to note that plasma levels of neutralizing Abs reached in clinical applications are well within or even higher than the concentration range tested in our in vitro setting (16, 17).

Because all Abs applied in our analyses were reported to neutralize IFN bioactivity, as confirmed by the manufacturer in cytopathic effect inhibition assays and as subsequently verified for the Fab and F(ab')2 fragments in our experiments, we investigated the combined application of rIFN and mAb on HDMECs. The full-length type I IFN blocking Abs were found to enhance rather than inhibit the endothelial response to IFN-{alpha} or IFN-β. In contrast, neutralizing Abs targeting IFN-{gamma} or the IFNRs IFNAR and IFNGR potently repressed IFIT-1 induction by rIFN. We have thus provided evidence that the ability of neutralizing Abs to elicit an IFN-like response or to further enhance the effects of type I IFN is only observed for mAbs targeting IFN-{alpha} or IFN-β in this cellular context.

Because the response was inhibited by concomitant administration of IFNAR blocking Abs even in the absence of exogenously added rIFN, we hypothesized that ECs might constitutively express low levels of type I IFNs which might contribute to the effects observed. The constitutive low-level expression of type I IFN has previously been reported for other cell types (1, 2). When testing EC culture supernatants, IFN was not detectable at ELISA sensitivities of 4 U/ml (IFN-{alpha}) or 10 U/ml (IFN-β). However, considering that basal subthreshold IFN concentrations were reported around 0.1 U/ml (2, 3), ELISA sensitivity may have been limiting (data not shown). We therefore proceeded to block the potential constitutive production of type I IFN by siRNA application. The approach was limited to IFN-β because silencing of IFN-{alpha} genes is difficult to accomplish due to the variety of IFN-{alpha} subtypes that may be expressed. Furthermore, basal expression of IFN-{alpha} in fibroblasts was shown to be dependent on the constitutive IFN-β production, arguing for a predominant role of IFN-β in the low level, basal expression of type I IFNs (18). Comparably, we observed that the application of IFN-β siRNA led to the concurrent down-regulation of IFN-β and IFN-{alpha} subtypes generally expressed in ECs (19). Silencing of IFN-β expression in HDMECs was found to greatly reduce the IFN-like response to IFN blocking mAbs: IFIT-1 expression levels were reduced to ~40% irrespective of the Ab specificity to IFN-{alpha} or IFN-β, thus reflecting the impact of IFN-β siRNA on the overall type I IFN expression. We therefore propose that ECs maintain a basal level of IFN expression and IFNAR phosphorylation, which allows for their IFN-like response to IFN blocking mAbs. Because the constitutive, weak IFN signal is also known to be a prerequisite for the efficient cellular response to a high-level IFN challenge in, for example, mouse embryonic fibroblasts (3), we conducted a control experiment with 100 pg/ml rIFN-{alpha} or rIFN-β. IFIT-1 induction by high-dose IFN was similarly impaired by IFN-β silencing. These results further support our argument for an essential, basal IFN-β expression in primary ECs, which promotes their capacity for efficient and rapid cellular activation.

The further investigations focused on the potential involvement of Fc domains in the endothelial activation by IFN-neutralizing Abs. As the mAbs tested were of the mouse IgG1 isotype, a cross-reaction with human Fc{gamma}Rs seemed feasible. When comparing the full-length Ab with Fab or F(ab')2 fragments of an IFN-{alpha} blocking mAb, the fragments did not elicit an IFN-like response in ECs thus pointing to the importance of the Ab Fc domain. Furthermore, the fragments potently inhibited IFIT-1 induction by rIFN, i.e., they exhibited the expected IFN-neutralizing capacity and they could compete for the activity of the corresponding full-length Ab. The latter was, however, not as effective as the inhibition of rIFN. This effect might potentially relate to a better accessibility of intact Ab to autocrine IFN if the Ab was membrane-associated, or bound to Fc receptors. The observation that the EC response to intact anti-IFN mAb was also reduced in the presence of a mouse IgG1 isotype Ab (containing an Fc portion), further suggested involvement of Fc receptors. ECs are known to express Fc{gamma}Rs with an apparent heterogeneity depending on the vessel type. Various isoforms of Fc{gamma}RII as well as Fc{gamma}RI and neonatal Fc receptor have been detected on ECs with CD32 being the most prominent on HDMECs (20, 21). Yet, we could not demonstrate CD32 expression on our endothelial isolates nor block the effect of the anti-IFN Abs by concomitant treatment with a neutralizing Ab directed against CD32 (data not shown). However, these results do not exclude the potential involvement of an endothelial Fc receptor other than CD32.

Interestingly, Fc receptors have been localized to EC caveolar membrane sections, which sets them in close proximity to IFNAR molecules (3, 22). With respect to their interrelation, two settings may be envisioned. The mere local proximity of IFNAR and Fc receptors might serve to sequester autocrine IFN at the cell surface. IFN-neutralizing Abs on Fc receptors might thus increase the local type I IFN concentration beyond the signal threshold provided that IFN bound to the blocking mAbs can be released, i.e., passed onto IFNAR. Alternatively, a direct receptor interaction between Fc receptor and IFNAR could occur, initiated by the anti-IFN-{alpha}/β Abs and resulting in IFNAR activation beyond the basal "ready state". Both players, IFNAR and Fc{gamma}Rs, have been reported to engage in diverse receptor cross-talk (2, 3, 23). Thus, whether Fc receptors are indeed involved in the IFN-like response to the neutralizing mAbs or whether the Ab Fc domain mediates engagement of another, as yet unidentified cellular factor, is of prime interest for further investigations.

The activating potential of neutralizing IFN Abs was observed for all types of ECs tested and was not abolished by concomitant proinflammatory activation of ECs. In addition, the fact that IFN-{alpha}/β-neutralizing Abs did not only trigger an IFN-like response on quiescent cells but could also enhance the effect of a high-dose IFN challenge, emphasizes the potentially adverse systemic implications. The induction level of IFN response genes varied to some extent with primary EC isolates. When other cell types were investigated, a heterogeneous response was observed that may relate to Fc repertoire or differences in the potency of the signaling cascade. Interestingly, the IFN-like response to monoclonal-neutralizing Abs directed against human type I IFN was also recorded for human PBMCs. Although this observation extends the potential clinical impact these Ab effects might have, it seems intriguing why these effects have not been noted previously by other groups in comparable experiments on leukocytes. In preliminary investigations, we have gathered an indication pointing to the importance of the Ab type. Although all the Abs presented in this study were monoclonals of the mouse IgG1 isotype, we did not find an IFN-like response to rabbit polyclonal anti-IFN antiserum in our experimental setting (data not shown). Thus, the nature and isotype of the Ab may be a crucial determinant.

The clinical application of recombinant mAbs targeted at IFN-{alpha} is at the current therapeutic focus of autoimmune diseases. In light of the recent launch of the first clinical trial testing an IFN-{alpha}-neutralizing mAb for the treatment of systemic lupus erythematosus, a possibly pleiotropic Ab effect would seem of particular concern. Our results would suggest that enhanced neutralizing efficiency might be achievable by testing Fab or F(ab')2 fragments vs full-length Igs in systemic settings.


   Acknowledgments
 
We thank R. Oehler and P. Petzelbauer for helpful suggestions and thorough evaluation of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant 7 from the Austrian Center of Excellence in Clinical and Experimental Oncology and by Grant P20074-B13 from the Austrian Science Fund. Back

2 H.P.M. and H.F. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Christine Brostjan, Medical University of Vienna, Surgery Department, Research Laboratories, Vienna General Hospital 8G9.13, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail address: Christine.Brostjan{at}meduniwien.ac.at Back

4 Abbreviations used in this paper: ISGF, IFN-stimulated gene factor; EC, endothelial cell; HDMEC, human dermal microvessel EC; IFIT-1, IFN-induced protein with tetratricopeptide repeats 1; IFNAR, IFN-{alpha} receptor; IFNGR, IFN-{gamma} receptor; ISG, IFN-stimulated gene; ISRE, IFN-stimulated response element; siRNA, small interfering RNA. Back

Received for publication February 9, 2008. Accepted for publication February 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Takaoka, A., H. Yanai. 2006. Interferon signalling network in innate defence. Cell. Microbiol. 8: 907-922. [Medline]
  2. Mitani, Y., A. Takaoka, S. H. Kim, Y. Kato, T. Yokochi, N. Tanaka, T. Taniguchi. 2001. Cross talk of the interferon-{alpha}/β signalling complex with gp130 for effective interleukin-6 signalling. Genes Cells 6: 631-640. [Abstract]
  3. Takaoka, A., Y. Mitani, H. Suemori, M. Sato, T. Yokochi, S. Noguchi, N. Tanaka, T. Taniguchi. 2000. Cross talk between interferon-{gamma} and -{alpha}/β signaling components in caveolar membrane domains. Science 288: 2357-2360. [Abstract/Free Full Text]
  4. Ogasawara, K., S. Hida, Y. Weng, A. Saiura, K. Sato, H. Takayanagi, S. Sakaguchi, T. Yokochi, T. Kodama, M. Naitoh, et al 2002. Requirement of the IFN-{alpha}/β-induced CXCR3 chemokine signalling for CD8+ T cell activation. Genes Cells 7: 309-320. [Abstract]
  5. Honda, K., T. Mizutani, T. Taniguchi. 2004. Negative regulation of IFN-{alpha}/β signaling by IFN regulatory factor 2 for homeostatic development of dendritic cells. Proc. Natl. Acad. Sci. USA 101: 2416-2421. [Abstract/Free Full Text]
  6. Ichikawa, E., S. Hida, Y. Omatsu, S. Shimoyama, K. Takahara, S. Miyagawa, K. Inaba, S. Taki. 2004. Defective development of splenic and epidermal CD4+ dendritic cells in mice deficient for IFN regulatory factor-2. Proc. Natl. Acad. Sci. USA 101: 3909-3914. [Abstract/Free Full Text]
  7. Selmi, C., A. Lleo, M. Zuin, M. Podda, L. Rossaro, M. E. Gershwin. 2006. Interferon {alpha} and its contribution to autoimmunity. Curr. Opin. Investig. Drugs 7: 451-456. [Medline]
  8. Stewart, T. A.. 2003. Neutralizing interferon {alpha} as a therapeutic approach to autoimmune diseases. Cytokine Growth Factor Rev. 14: 139-154. [Medline]
  9. Chuntharapai, A., J. Lai, X. Huang, V. Gibbs, K. J. Kim, L. G. Presta, T. A. Stewart. 2001. Characterization and humanization of a monoclonal antibody that neutralizes human leukocyte interferon: a candidate therapeutic for IDDM and SLE. Cytokine 15: 250-260. [Medline]
  10. Brostjan, C., A. Bayer, A. Zommer, A. Gornikiewicz, S. Roka, T. Benko, R. Yaghubian, R. Jakesz, G. Steger, M. Gnant, et al 2003. Monitoring of circulating angiogenic factors in dendritic cell-based cancer immunotherapy. Cancer 98: 2291-2301. [Medline]
  11. Wang, X., B. Seed. 2003. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 31: e154[Abstract/Free Full Text]
  12. Brostjan, C., T. Bellón, Y. Sobanov, M. López-Botet, E. Hofer. 2002. Differential expression of inhibitory and activating CD94/NKG2 receptors on NK cell clones. J. Immunol. Methods 264: 109-119. [Medline]
  13. Brostjan, C., J. Anrather, V. Csizmadia, G. Natarajan, H. Winkler. 1997. Glucocorticoids inhibit E-selectin expression by targeting NF-{kappa}B and not ATF/c-Jun. J. Immunol. 158: 3836-3844. [Abstract]
  14. Brostjan, C., J. Anrather, V. Csizmadia, D. Stroka, M. Soares, F. H. Bach, H. Winkler. 1996. Glucocorticoid-mediated repression of NF-{kappa}B activity in endothelial cells does not involve induction of I{kappa}B{alpha} synthesis. J. Biol. Chem. 271: 19612-19616. [Abstract/Free Full Text]
  15. Hernández, J. L., T. Coll, C. J. Ciudad. 2004. A highly efficient electroporation method for the transfection of endothelial cells. Angiogenesis 7: 235-241. [Medline]
  16. Gottlieb, A. B., S. Masud, R. Ramamurthi, A. Abdulghani, P. Romano, U. Chaudhari, L. T. Dooley, A. A. Fasanmade, C. L. Wagner. 2003. Pharmacodynamic and pharmacokinetic response to anti-tumor necrosis factor-{alpha} monoclonal antibody (infliximab) treatment of moderate to severe psoriasis vulgaris. J. Am. Acad. Dermatol. 48: 68-75. [Medline]
  17. Margolin, K., M. S. Gordon, E. Holmgren, J. Gaudreault, W. Novotny, G. Fyfe, D. Adelman, S. Stalter, J. Breed. 2001. Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: pharmacologic and long-term safety data. J. Clin. Oncol. 19: 851-856. [Abstract/Free Full Text]
  18. Erlandsson, L., R. Blumenthal, M. L. Eloranta, H. Engel, G. Alm, S. Weiss, T. Leanderson. 1998. Interferon-β is required for interferon-{alpha} production in mouse fibroblasts. Curr. Biol. 8: 223-226. [Medline]
  19. Lagos, D., M. W. Trotter, R. J. Vart, H. W. Wang, N. C. Matthews, A. Hansen, O. Flore, F. Gotch, C. Boshoff. 2007. Kaposi sarcoma herpesvirus-encoded vFLIP and vIRF1 regulate antigen presentation in lymphatic endothelial cells. Blood 109: 1550-1558. [Abstract/Free Full Text]
  20. Devaraj, S., B. Davis, S. I. Simon, I. Jialal. 2006. CRP promotes monocyte-endothelial cell adhesion via Fc{gamma} receptors in human aortic endothelial cells under static and shear flow conditions. Am. J. Physiol. 291: H1170-H1176.
  21. Groger, M., G. Sarmay, E. Fiebiger, K. Wolff, P. Petzelbauer. 1996. Dermal microvascular endothelial cells express CD32 receptors in vivo and in vitro. J. Immunol. 156: 1549-1556. [Abstract]
  22. Mineo, C., P. W. Shaul. 2006. Circulating cardiovascular disease risk factors and signaling in endothelial cell caveolae. Cardiovasc. Res. 70: 31-41. [Abstract/Free Full Text]
  23. Ortiz-Stern, A., C. Rosales. 2003. Cross-talk between Fc receptors and integrins. Immunol. Lett. 90: 137-143. [Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moll, H. P.
Right arrow Articles by Brostjan, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moll, H. P.
Right arrow Articles by Brostjan, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS