A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

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  • A monoclonal antibody and an enzyme immunoassay

    for human Ala-IL-877

    Natalia N. Nashkevicha, Svetlana Akalovicha, Natalia Lounevab,George A. Heavnerc, Nikolai N. Voitenoka,d,*

    aLaboratory of Cellular and Molecular Immunology, Institute of Hematology and Blood Transfusion, Dolginovsky Tract 160, Minsk, BelarusbByelorussian Institute for Hereditary Diseases, Minsk, Belarus

    cCentocor Inc., Malvern, PA, USAdFund for Molecular Hematology and Immunology, Post Box 338, Moscow 125493, Russia

    Received 4 February 2002; received in revised form 30 April 2002; accepted 21 June 2002

    Abstract

    Interleukin-8 (IL-8) plays a central role in neutrophil chemotaxis and exerts a wide range of effects on various cells, ranging

    from tumor angiogenesis to impairment of neuronal signaling. Two main forms of IL-8 exist, one containing 77 amino acids

    (Ala-IL-877) and a second containing 72 amino acids (Ser-IL-872), which comprise more than 90% of IL-8 protein in cell

    cultures. IL-877 was reported to be produced predominantly by endothelial cells and is known as endothelial IL-8. IL-872predominates in monocyte cultures and is known as leukocyte IL-8. While both forms have equal chemotactic activity in

    vivo, recent data suggest that their biological activities might be different. Here we describe the generation of a mouse

    monoclonal antibody (mAb) specific for IL-877 and the development of a corresponding immunoassay. Various immunization

    protocols were investigated. Immunization with conjugates of a peptide from the N-terminus of IL-877 (NTP77) resulted in the

    production of an IgG1 mAb (N11) that recognizes human IL-877 and neutralizes its chemotactic activity. A sensitive ELISA

    specific for IL-877 was developed using N11 for capture and a biotinylated mAb to IL-872 for detection. Using this

    immunoassay it was shown that the only form of IL-8 secreted in cell culture was IL-877 and that the IL-872 present was the

    result of proteolysis of IL-877. IL-877 was detected in plasma and cerebrospinal fluid (CSF) from patients with sepsis and

    meningitis.

    D 2002 Elsevier Science B.V. All rights reserved.

    Keywords: Ala-interleukin-8; Monoclonal antibody; ELISA; Culture supernatants; Septic plasma

    0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

    PII: S0022 -1759 (02 )00279 -X

    Abbreviations: IL-8, interleukin-8; mAb, monoclonal antibody; Ig, immunoglobulin; NTP77, N-terminal peptide of IL-877; CFA/IFA,

    complete/incomplete Freunds adjuvant; i.p., intraperitoneally; BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; Ova,

    ovalbumin; Sulfo-MBS, m-maleimidobenzoyl sulfosuccinimide ester; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; TNF-a, tumornecrosis factor-a; IL-1-h, interleukin-1-h; LPS, lipopolysaccharide; PI, protease inhibitors cocktail; CSF, cerebrospinal fluid.

    * Corresponding author. Laboratory of Cellular and Molecular Immunology, Institute of Hematology and Blood Transfusion, Dolginovsky

    Tract 160, 223059 Minsk, Belarus. Tel.: +7-375-172-344-483; fax: +7-375-172-310-472.

    E-mail address: nvoitenok@infonet.by (N.N. Voitenok).

    www.elsevier.com/locate/jim

    Journal of Immunological Methods 270 (2002) 3751

  • 1. Introduction

    Interleukin-8 (IL-8) belongs to the CXC family of

    chemokines (Baggiolini, 2001) and plays a central

    role in neutrophil chemotaxis and activation (Baggio-

    lini et al., 1994). In addition, IL-8 exerts pleiotropic

    actions (Mukaida, 2000) including angiogenesis (Bel-

    perio et al., 2000), alteration of neuronal signaling

    (Giovannelli et al., 1998) and leukemic cell apoptosis

    (Terui et al., 1998). IL-8 is produced by multiple cell

    types under the influence of bacterial products, in

    response to inflammatory cytokines such as tumor

    necrosis factor and interleukin-1, or after malignant

    transformation (Matsushima and Oppenheim, 1989;

    Baggiolini et al., 1994; Mukaida, 2000).

    IL-8 secreted by cultured monocytes showed het-

    erogeneity in the N-terminal sequence and the amino

    acid length of the IL-8 protein ranged from 79 amino

    acids through 77, 72, 71, 70 to a 69-amino-acid

    variant (Lindley et al., 1988; Van Damme et al.,

    1988; Yoshimura et al., 1989; Schroder et al., 1990).

    Two main forms of IL-8 containing 77 or 72 amino

    acids were termed Ala-IL-877 (IL-877) and Ser-IL-872(IL-872), respectively, and account for more than 90%

    of the IL-8 in monocyte culture (Yoshimura et al.,

    1989). The various forms of IL-8 are generated via

    proteolytic cleavage of the primary 99-amino-acid

    precursor following cleavage of the 20-amino-acid

    signal sequence (Yoshimura et al., 1989; Baggiolini

    et al., 1994). IL-877 was reported to be produced

    predominantly by endothelial cells and is known as

    endothelial IL-8 (Gimbrone et al., 1989; Hebert et

    al., 1990; Huber et al., 1991) while IL-872 is known as

    leukocyte-derived IL-8 (Hebert et al., 1990; Bag-

    giolini et al., 1994). While IL-872 is a more potent

    neutrophil chemoattractant when tested by in vitro

    assays (Hebert et al., 1990; Nourshargh et al., 1992),

    both forms have equal in vivo neutrophil chemotactic

    activity (Nourshargh et al., 1992) attributed to the

    GluLeuArg (ELR) motif (amino acids 6967)

    (Baggiolini et al., 1994).

    Recent data suggest that the biological activity of

    IL-877 may differ from that of IL-872 in terms of its

    capacity to induce apoptosis of leukemic cell lines

    (Terui et al., 1998, 1999).

    The production and role of IL-877 and IL-872 in

    inflammatory responses and pathology in vivo are

    still obscure, since existing immunoassays are unable

    to distinguish the two molecules. N-terminal amino

    acid sequencing and molecular size estimation by

    polyacrylamide gel electrophoresis (PAGE) were

    used for the identification of IL-8 forms in cell

    cultures (Lindley et al., 1988; Van Damme et al.,

    1988; Yoshimura et al., 1989) and in vivo (Renne-

    kampff et al., 2000).

    In this report we describe the production of a

    monoclonal antibody (mAb) to IL-877 and the devel-

    opment of a specific enzyme immunoassay. This

    ELISA was able to quantify IL-877 in cell cultures,

    blood plasma and cerebrospinal fluid (CSF) obtained

    from patients with sepsis and meningitis.

    2. Materials and methods

    2.1. Animals and reagents

    Female BALB/c mice (BALB/cJCitMoise) of 10

    12 weeks of age were obtained from the breeding

    nucleus of the Shemyakin & Ovchinnikov Institute of

    Bioorganic Chemistry, Moscow. Mice were housed in

    accordance with institutional guidelines.

    Recombinant human IL-877 and IL-872 (r-IL-8)

    were obtained from Peprotech, Rocky Hill, NJ, or

    kindly donated by Dr. Ji Ming Wang (Lab. Molecular

    Immunoregulation, NCI at Frederick, Frederick, MD).

    Synthetic IL-877 was kindly donated by Prof. Marco

    Baggiolini, Kocher Inst., University Bern. The peptide

    AVLPRSAKELRC-NH2 (NTP77) corresponding to

    the N-terminal part of human IL-877 was synthesized

    by the Protein Design group at Centocor, Malvern, PA

    on Rink resin using an ABI 431A Synthesizer with the

    standard single coupling FMOC protocol. After cleav-

    age using a cocktail of trifluoroacetic acid (TFA),

    phenol, dithiothreitol, thioanisole and water, the pep-

    tide was purified by reverse phase (C-18) HPLC using

    a linear gradient of 3080% acetonitrile in 0.1% TFA.

    The purified peptide had the correct amino acid

    analysis and mass spectra. The N-terminal pentapep-

    tide of IL-877, AVLPR, was synthesized by PerSeptive

    Biosystems, Department of Microbiology and Immu-

    nology, University of Maryland, School of Medicine,

    Baltimore, MD. Recombinant human IL-877 fused to

    human FcIgG (FcIgG IL-877) and recombinant

    human tumor necrosis factor-a (TNF-a) receptor p-55 fused to human FcIgG (FcIgGp-55) (Scallon et

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375138

  • al., 1995) were kindly provided by Dr. Bernard

    Scallon, Centocor. The N-terminal peptide corre-

    sponding to the N-terminal 22 amino acids of the

    human IL-8 receptor CXCR2 was kindly donated by

    Dr. Ernst Brandt (Forschungszentrum, Borstel, Ger-

    many). Rabbit polyclonal antibody to human IL-8 was

    kindly donated by Prof. Sergei Ketlinsky (Inst. Ultra-

    pure Biopreparates, St. Petersburg, Russia). Complete

    (CFA) and incomplete (IFA) Freunds adjuvants were

    from Sigma, St. Louis, MO. RIBI adjuvant MPL-

    TDM system was from RIBI ImmunoChem Research,

    Hamilton, MT.

    2.2. Conjugation protocol

    Bovine serum albumin (BSA) (Pierce, Rockford,

    IL), ovalbumin (Ova) and keyhole limpet hemocyanin

    (KLH) (both from Sigma) were used for peptide

    conjugation. The peptide AVLPRSAKELRC-NH2,

    corresponding to the N-terminal sequence of IL-877,

    was conjugated to carrier proteins in two ways: (1)

    adding sulfhydryl groups to the peptide with 2-imi-

    nothiolane (Jue et al., 1978; Aithal et al., 1988)

    followed by conjugation to BSA, Ova, or KLH using

    the heterobifunctional agent m-maleimidobenzoyl sul-

    fosuccinimide ester (sulfo-MBS) (Kitagawa et al.,

    1982); or, (2) by water-soluble carbodiimide in a

    single-step coupling reaction (Catty and Raykundalia,

    1988).

    2.3. Immunization

    BALB/c mice were immunized intraperitoneally

    (i.p.) on the splenic side with 25 or 50 Ag ofrecombinant human FcIgGIL-877 fusion protein or

    NTP77 conjugated to BSA, Ova or KLH in 100 Al ofphosphate-buffered saline (PBS) emulsified in 100 Alof CFA. Mice were boosted i.p. on the contralateral

    side at monthly intervals with 2550 Ag of recombi-nant human FcIgGIL-877 fusion protein, 2550 Agof NTP77 conjugate or 2030 Ag of synthetic IL-877in 100 Al of saline solution emulsified at 1:1 with IFA.Blood samples were collected from the tail vein of

    anaesthetized mice on day 20 after each immuniza-

    tion. Each group consisted of three or more mice.

    Serial dilutions of mouse sera in PBS with 1.0% BSA

    were assayed in triplicate in 96-well polystyrene

    plates (Corning Inc., Corning, NY) coated with

    NTP77, IL-877, IL-872 or NTP77 conjugates at 1 Ag/ml and developed with goat anti-mouse horseradish

    peroxidase conjugate (Bio-Rad, Richmond, CA). Titer

    was defined as the last dilution, which yielded a value

    above the negative control (normal mouse sera). The

    levels of IL-8 specific antibodies in mouse sera were

    expressed as relative units (RU) against the standard

    mAb WS-4 at 1 Ag/ml.

    2.4. Monoclonal antibodies: generation and purifica-

    tion

    Spleen cells were fused to the myeloma cells

    P3X63-Ag8.653 and cloned as previously described

    (Lane et al., 1984; Panyutich et al., 1991). Hybridoma

    clones producing antibodies were identified by ELISA

    using microtiter plates coated with free NTP77, NTP77conjugates, corresponding carriers, IL-877, IL-872 or

    FcIgGIL-877 fusion protein as indicated in Table 1

    and were developed with peroxidase-labeled goat

    anti-mouse antibody. Selected hybridoma wells were

    subcloned twice by limiting dilution over a BALB/c

    peritoneal macrophage feeder layer. The IgG subclass

    of the mAbs was determined using an isotyping kit

    from Fisher Biotech (Pittsburgh, PA). The resulting

    mAbs were produced by injecting hybridoma cells i.p.

    into BALB/c mice primed with pristane (Sigma). For

    in vitro analysis, mAbs were purified from hybridoma

    supernatants or ascites by Protein A or Protein G

    chromatography (Pharmacia Fine Chemicals, Piscat-

    away, NJ). Protein concentrations were determined by

    the Bradford dye binding assay (Bio-Rad).

    2.5. Characterization of monoclonal antibodies to

    IL-8

    2.5.1. Hybridomas origin and isotyping data

    Hybridomas specific for IL-872 (4C, 3A, N6) and

    anti-IgG clone C12 were derived from mice immu-

    nized with recombinant proteins FcIgGIL-877 and IL-

    8. The previously described anti-IL-872 hybridoma

    clonesWS-4 and BS-1 (Ko et al., 1992) and hybridoma

    H6 were from our internal panel. Hybridoma H9,

    specific for the sulfo-MBS-link, was derived from

    sequential immunization with BSANTP77 and

    OvaNTP77 (sulfo-MBS coupled). N11 and D5 clones

    were derived from immunization with KLHNTP77(sulfo-MBS coupled) followed by BSANTP77 (car-

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 39

  • bodiimide conjugate) and synthetic IL-877. All these

    hybridoma clones produced IgG1, n isotype antibod-ies, except the clone D5 that produced an IgM mAb.

    2.5.2. Chemotaxis assay

    Neutrophil chemotaxis was performed against

    recombinant IL-877 and IL-872 using 48-well micro

    Boydens chambers as previously described (Falk et

    al., 1980). Briefly, binding buffer containing 1% BSA

    in RPMI 1640 with or without mAb (at 0.550 Ag/ml)and IL-877 or IL-872 (at 10 ng/ml) was placed in the

    lower compartment of the chamber. A 5-Am polycar-bonate filter (Neuroprobe, Cabin John, MD) was

    placed at the intersection of the upper and lower

    compartments. Freshly isolated neutrophils (106

    cells/ml) in binding medium were added to the upper

    compartment of the chamber. After incubation for 2 h

    at 37 jC, the upper surface of the filter was scrapedand the resulting cells fixed with methanol and stained

    with Leukostat (Fisher Scientific). Data were analyzed

    using the BIOQUANT program (R & M Biomatrics,

    Nashville, TN) and the results were expressed as the

    mean number of migrated cells per 10 fields at 10magnification.

    2.5.3. Immunoblots

    SDS-PAGE was performed by the Laemmli proce-

    dure using 20% acrylamide separation gels. Samples

    were electrotransferred from gels to nitrocellulose

    membranes (Hybond C) and incubated with mAb

    N11, 4C or WS-4 at 10 Ag/ml, followed by reactionwith the alkaline phosphatase-labeled goat anti-mouse

    IgG antibody (Bio-Rad) and developed using NBT/

    BCIP as a substrate.

    2.5.4. Immunohistochemistry

    Mononuclear leukocytes isolated from the fresh

    heparinized blood of healthy donors using standard

    Ficoll/Hypaque protocol were incubated for 3.5 h in

    60 mm plastic dishes (Corning Glass Works, Corning,

    NY) at 37 jC in RPMI5% FCS (Sigma) in thepresence of 100 ng/ml lipopolysaccharide (LPS) from

    E. coli and brefeldin at 10 Ag/ml (both from Sigma).Cell smears were fixed in ice-cold acetone for 10 min

    or in neutral buffered 10% formalin solution for 10

    min and stained with mAb N11, WS-4 or an isotype

    control mouse IgG1 mAb at 10 Ag/ml overnight at 4jC, followed by treatment with horseradish peroxi-dase-conjugated rabbit anti-mouse antibody (Dako) or

    an LSAB kit (Dako) according to the manufacturers

    protocol. All steps of staining of formalin-fixed

    smears were performed in the presence of 0.01%

    saponin (Sigma).

    2.6. ELISA detecting IL-8

    The selection of mAb pairs for the detection of IL-

    877 and optimization of an IL-877 ELISA was per-

    formed using the basic protocol described below.

    Microtiter plates were coated with 5 Ag/ml of thecapture mAb in 100 Al of 0.1 M sodium carbonatebuffer, pH 9.6 or PBS, pH 7.4 overnight at 4 jC.After washing the plates three times with 0.3 M NaCl

    in PBS, pH 7.4 and 0.05% Tween-20 (PBS0.3 M

    NaClTw), unbound sites were blocked by adding

    Table 1

    Specificity of mAbs generated after immunization with FcIgG

    IL-877 or NTP77 conjugates

    Antigen Absorbance (492 nm)

    Antibodies

    4C C12 H9 N11 D5

    IL-872 2.717 0.045 0.186 0.015 0.237

    IL-877 2.717 0.038 0.221 2.481 2.367

    NTP77 0.030 0.046 0.216 2.153 0.025

    BSANTP77a 0.040 0.051 2.207 2.419 0.051

    BSANTP77b 0.039 n.t.c 0.166 2.239 0.105

    BSA 0.020 0.050 0.160 0.012 0.023

    OvaNTP77a 0.028 0.080 2.328 2.246 0.021

    Ova 0.019 0.063 0.221 0.012 0.025

    FcIgGIL-877 2.595 1.624 0.196 1.797 1.451

    Human IgG 0.036 1.000 0.267 0.019 0.028

    FcIgGp55d 0.052 1.210 0.268 0.020 0.035

    KLHNTP77a 0.015 n.t. 2.223 1.232 0.028

    KLH 0.021 n.t. 0.152 0.010 0.025

    KLHCXCR2 NTPa,e n.t. n.t. 1.927 0.015 n.t.

    BSAAVLPRb n.t. n.t. n.t. 0.025 n.t.

    Microtiter plates were coated with 0.1 Ag of each antigen per welland tested against hybridoma clone supernatants diluted 1/5 with

    PBS0.5% BSA0.05% Tween-20, followed by development with

    peroxidase-labeled goat anti-mouse antibody as described in the

    Materials and Methods. Means of duplicates are shown. Absorb-

    ency data considered as positive recognition are printed in bold.a Conjugation with sulfo-MBS.b Conjugation with carbodiimide.c n.t.non-tested.d Recombinant protein consisting of human TNF-R p55 fused to

    FcIgG.e Hemocyanin conjugated via sulfo-MBS with N-terminal

    peptide (22 amino acids) of CXCR2 receptor of IL-8.

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375140

  • 0.5% BSA in PBS0.3 M NaClTw at room temper-

    ature (RT) for 1 h. IL-872, IL-877 (stored at 10 Ag/mlin PBS1% BSA at 70 jC) and samples wereserially diluted in PBS0.3 M NaCl0.5% BSATw,

    added to the wells (in triplicate) and incubated at RT

    for 1 h. Plates were washed three times with PBS0.3

    M NaClTw after each subsequent step. Antibodies

    used in the liquid phase were labeled with biotin

    using Sulfo-NHS-LC-Biotinylation Kit (Pierce, Rock-

    ford, IL) and added at 0.51 Ag/ml for 1 h at RT.After washing, the plates were incubated with horse-

    radish peroxidase-conjugated avidin. Plates were

    washed and developed with 0.4 mg/ml o-phenylene-

    diamine (Sigma) in 20 mM citrate buffer, pH 4.7

    containing 0.01% H2O2. After 10 min, the reaction

    was quenched with 5% H2SO4 and absorbance values

    were read at 492 nm. When a rabbit antibody to IL-8

    was used for the liquid phase, plates were developed

    with an alkaline phosphatase-labeled goat anti-rabbit

    IgG (Boehringer Mannheim, Germany) using p-nitro-

    phenylphosphate (Labsystems, Helsinki, Finland)

    and read at 405 nm.

    2.7. Induction of IL-8 in cell cultures

    Mononuclear leukocytes (MNL) in RPMI10%

    FCS, isolated from fresh heparinized blood using the

    standard Ficoll/Hypaque protocol, were incubated in

    24-well plastic plates (Corning Glass Works) for 1 h at

    37 jC as previously described (Chaly et al., 2000).Adherent MNL were incubated in RPMI2% FCS in

    the presence of 100 ng/ml LPS. At different time

    points, supernatant was aspirated and stored frozen or

    at 4 jC with or without a protease inhibitor cocktail(PI) (Protease inhibitor cocktail tablets Complete,

    Mini, Roche Diagnostics, Mannheim, Germany). Bre-

    feldin (10 Ag/ml) was added to some of the cultures 15min before the addition of LPS. Supernatants from

    human breast adenocarcinoma cell lines, cultured in

    either serum-free or serum-containing media were

    kindly provided by Dr. Rosalba Salsedo, Lab. Molec-

    ular Immunoregulation, FCDRC, Frederick, MD.

    Neuroblastoma cell lines SK-N-MC (ATCC HTB

    10) and SK-N-SH (ATCC HTB 11) were obtained

    from ATCC (Rockville, MD) and cultured in IMDM

    10% FCS. Human umbilical vein endothelial cells

    (HUVEC) were purchased from Clonetics (Walker-

    ville, MD) and subcultured according to the manu-

    facturers instructions in Endothelial Cell Growth

    Medium (EGM, Clonetics). Neuroblastoma cells and

    HUVEC were stimulated for 20 h with either human

    TNF-a at 510 ng/ml or interleukin-1-h (IL-1-h)(both Peprotech) at 5 ng/ml.

    2.8. Detection of IL-877 in clinical samples

    Blood and CSF samples were obtained from the

    Dept. of Intensive Care Medicine of the Pediatric

    Infections City Hospital, Minsk, and Urgent Medicine

    Hospital, Minsk. Blood samples were anticoagulated

    with EDTA (5 mM final concentration) and mixed

    with the protease inhibitor cocktail at the recommen-

    ded concentration. Samples were immediately centri-

    fuged and the plasma separated and stored at 40 or 70 jC. Cerebrospinal fluid samples were preservedwith PI and stored frozen at 40 jC.

    3. Results

    3.1. Serum antibody responses to FcIgGIL-877 and

    conjugates of NTP77

    Mouse sera were screened for their ability to bind

    NTP77 or r-IL-872. Since the fusions of spleens from

    more than 20 selected animals immunized with

    FcIgGIL-877 or BSANTP77 failed to produce a

    single mAb specific for IL-877, we briefly present our

    observations on antibody responses and an immuni-

    zation protocol that resulted in the generation of mAbs

    specific for IL-877.

    The use of 50 Ag FcIgGIL-877 fusion protein inCFA for priming followed by boosting with 25 Ag ofFcIgGIL-877 in IFA resulted in an approximately

    three-fold greater antibody response to IL-8 as

    compared to the same sensitization using free IL-8

    (Fig. 1). Freunds adjuvant with FcIgGIL-877yielded about 2.5-fold higher anti-IL-8 antibody

    levels when compared with RIBI adjuvant (not

    shown). We were unable to boost mice with 50 AgFcIgGIL-877 in IFA since gross lesions and mor-

    bidity were observed in mice primed with 50 Ag ofFcIgGIL-877 in CFA and boosted with the same

    dose of FcIgGIL-877 in IFA. No morbidity or

    significant lesions were present in mice boosted with

    25 Ag of FcIgGIL-877 in IFA.

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 41

  • Surprisingly, the second boost with FcIgGIL-877in IFA failed to stimulate an anti-IL-8 response (Fig.

    1), while antibody titers to human IgG increased (not

    shown). In contrast, the use of r-IL-8 in IFA for the

    second boost instead of FcIgGIL-877 resulted in

    more than a three-fold increase of anti-IL-8 antibody

    levels (Fig. 1). Interestingly, when r-IL-8 was used for

    the first boost, antibody levels were two-fold less

    (Fig. 1).

    Mice immunized using the mixed FcIgGIL-877/r-

    IL-8 immunization protocol (Fig. 1) showed higher

    antibody titers to IL-872 compared to NTP77 (about

    20:1, data not shown). More than 10 fusions of

    spleens from these mice failed to produce NTP77specific mAbs, while numerous hybridomas to IL-872or human IgG were generated (4C, C12, Table 1).

    We primed a group of mice with 50 Ag of BSANTP77 conjugate in CFA followed by two boosts with

    the same antigen in IFA at monthly intervals. As in the

    case of sensitization with FcIgGIL-877, the second

    boost with the BSANTP77 conjugate gave no higher

    antibody titers to NTP77 (not shown). However, when

    OvaNTP77 conjugate was used for the second boost,

    an anti-NTP77 response that was about 3.5-fold

    greater was observed (not shown). Nevertheless,

    fusions of spleens from selected mice from this

    protocol failed to produce a single anti-NTP77 hybrid-

    oma, while mAbs reacting with irrelevant conjugates

    coupled through a sulfo-MBS-link were identified

    (H9, Table 1), indicating that the sulfo-MBS bond

    region itself induced a specific antibody response after

    the priming and two boosts. Immunization with sulfo-

    MBS coupled KLHNTP77 followed by sequential

    boosting with carbodiimide conjugated BSANTP77and synthetic IL-877 gave two NTP77-specific hybrid-

    omas in the first spleen fusion (N11 and D5) as shown

    in Table 1.

    3.2. Specificity of monoclonal antibodies derived from

    mice immunized with FcIgGIL-877 and NTP77conjugates

    Multiple mAbs to IL-872 (4C, Table 1, 3A and N6,

    Fig. 6) or human IgG (C12, Table 1) were produced

    by fusions of splenocytes from mice immunized with

    FcIgGIL-877. Antibody 4C did not react with NTP77(Table 1) which contained the chemotactic ELR motif

    but competed with N11 mAb for IL-877 binding (data

    not shown). It was the most efficient mAb in neutral-

    izing IL-8 among those available for comparison (data

    not shown), including mAb WS-4 (Ko et al., 1992).

    Similar to WS-4 (Harada et al., 1993), 4C cross-

    reacted with rabbit IL-8 (data not shown). Each

    mAb capable of binding to the 72-amino-acid form

    of IL-8 also recognized IL-877 (Table 1).

    Hybridoma H9 was derived from mice immunized

    with sulfo-MBS coupled conjugates of NTP77 with

    BSA and Ova. This mAb recognized sulfo-MBS

    coupled BSANTP77 and an irrelevant conjugate of

    KLH and the N-terminal peptide of human CXCR2

    but did not react with carbodiimide conjugated BSA

    NTP77, indicating sulfo-MBS-link specificity.

    Monoclonals D5 and N11 showed specificity for

    the 77-amino-acid form of IL-8 and were derived

    from the mouse immunized with sulfo-MBS coupled

    KLHNTP77 followed by carbodiimide conjugated

    BSANTP77 and synthetic IL-877. D5 reacted with

    IL-877 and FcIgGIL-877 but did not react with

    NTP77 and showed weak recognition of IL-872. In

    contrast, mAb N11 reacted with IL-877, FcIgGIL-877

    Fig. 1. Comparison of the antibody response to human IL-8 after the

    first and the second boost with FcIgGIL-877 or recombinant IL-8.

    On day 0, groups of three mice were primed with FcIgGIL-877 in

    CFA and boosted on day 30 and on day 60 with FcIgGIL-877 (n)or r-IL-8 (o) in IFA. Blood for titration was taken on day 20 aftereach immunization. Sera were assayed for IL-8 specific antibodies

    by serial dilutions in an ELISA and titers expressed as relative units

    against the standard mAb WS-4 at 1 Ag/ml. Values are shown forthe meanF S.E. (n= 3). Mice primed with r-IL-8 showed less than1.5 RU after the first boost with r-IL-8 in Freunds adjuvant (not

    shown).

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375142

  • and free or conjugated NTP77 but did not recognize

    IL-872. This mAb was selected for further study.

    3.3. Characterization of antibody N11

    3.3.1. Specificity of mAb N11 in ELISA

    The specificity of N11 adsorbed on polystyrene

    plates was characterized using a liquid phase poly-

    clonal antibody to IL-8 in an ELISA format. IL-877showed concentration-dependent binding to N11 (Fig.

    2A), while no binding of IL-872 was seen. Both forms

    of IL-8 bound to the BS-1 mAb that recognized IL-872(Fig. 2B).

    IL-877 is converted to IL-872 by cleavage of the

    AVLPR peptide with serine proteases at the ArgUSerbond (Yoshimura et al., 1989). We explored the

    possibility that the peptide AVLPR could inhibit bind-

    ing of IL-877 to N11. Fig. 3 shows that the AVLPR

    peptide was capable of inhibiting the binding of N11

    to IL-877 and NTP77 but a 103 molar excess of

    AVLPR over IL-877 was needed to obtain a significant

    inhibition. The binding of N11 to NTP77 was more

    sensitive to inhibition by AVLPR (Fig. 3) suggesting

    that the affinity of N11 to NTP77 was much lower than

    to IL-877. Interestingly, N11 mAb was unable to bind

    a solid phase BSAAVLPR conjugate (Table 1) and

    free AVLPR peptide (not shown).

    3.3.2. Characterization of N11 using immunoblotting

    and cell immunostaining

    The ability to recognize antigens after denaturing

    PAGE and in fixed cell and tissue preparations repre-

    sents an important characteristic of mAbs to cytokines.

    Antibody N11 recognized IL-877 and conjugates of

    NTP77 on immunoblots after Laemmli denaturing

    PAGE, while IL-872 was not recognized (data not

    shown). As expected, mAbs 4C and WS-4 recognized

    both forms of IL-8. When tested in immunohistolog-

    ical staining of human mononuclear leukocytes acti-

    vated with LPS for 3.5 h in the presence of brefeldin,

    N11 showed efficient immunostaining of cell smears

    fixed with ice-cold acetone or formalin/0.01% saponin

    (data not shown).

    Fig. 2. Comparison of the anti-IL-877 mAb N11 (A) and the anti-IL-

    872 mAb BS-1 (B) used as capture antibodies in the ELISA.

    Microtiter plates were coated with mAbs (5 Ag/ml) and incubatedwith IL-877 or IL-872 as described in the Materials and Methods. A

    rabbit anti-IL-8 antibody was used for the liquid phase, followed by

    an alkaline phosphatase-labeled goat antibody to rabbit IgG. Means

    of triplicates are shown.

    Fig. 3. Inhibition of binding of the anti-IL-877 mAb N11 or the anti-

    IL-872 mAb H6 to plastic adsorbed IL-877 or NTP77 in the presence

    of increasing concentrations of the AVLPR peptide or NTP77.

    Microtiter plates were coated with 0.1 Ag/ml of r-IL-877 (.) orNTP77 (5) and incubated with mAb N11 or the anti-IL-872 mAb H6in the presence of increasing concentrations of AVLPR ( +AVLPR)

    or NTP77 ( +NTP77) and were developed with peroxidase-labeled

    goat anti-mouse antibody.

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 43

  • 3.3.3. N11 mAb is efficient in homologous ELISA

    Dimeric and oligomeric forms of proteins in sol-

    ution can be recognized by so-called oligomeric or

    homologous (hm) ELISA, which uses the same

    mAb specific for a single epitope on a monomeric

    protein for both the solid and the liquid phase (Corti et

    al., 1992; Petyovka et al., 1995). In a homologous

    ELISA, N11 recognized IL-877 in PBS0.5% BSA at

    concentrations ranging from 2 to 16 ng/ml (Fig. 4A),

    demonstrating that IL-877 is a dimer (or oligomer) at

    physiologically relevant concentrations. The non-

    ionic detergent Tween-20 dissociated IL-877 to mono-

    mers that were not recognized by N11/N11 hm-

    ELISA (Fig. 4A), but were recognized in a heterol-

    ogous ELISA consisting of captured N11 and bio-

    tinylated H6 (Fig. 4B). These results are similar to

    those obtained for the oligomeric forms of IL-872(Petyovka et al., 1995) and TNF-a (Corti et al.,1992; De Groote et al., 1993; Petyovka et al., 1995).

    3.3.4. Antibody N11 neutralizes chemotactic activity

    of IL-877Antibody N11 completely inhibited chemotactic

    activity of IL-877, while chemotactic activity of IL-872was not affected in the presence of increasing concen-

    trations of N11 (Fig. 5A). As expected, the mAb 4C to

    IL-872 inhibited chemotactic activity of both forms of

    IL-8 (Fig. 5B).

    Fig. 4. Titration of IL-877 in a homologous ELISA (A) using N11

    mAb for capture and biotinylated N11 for detection (N11/N11*),

    compared to a heterologous ELISA (B) for IL-877 consisting of N11

    for capture and biotinylated H6 (N11/H6*) for detection in the

    presence (.) or in the absence (o) of 0.05% Tween-20. * Representsbiotinylated mAb, the same as in Figs. 6 and 7.

    Fig. 5. The effect of the anti-IL-877 mAb N11 (A) and the anti-IL-872mAb 4C (B) on the human neutrophil chemotactic activity of IL-877(.) and IL-872 (o). Spontaneous cell migration differed, but wastypically in the range of 1530 cells per high power field (No./HPF).

    The mean and F S.E. of three experiments are shown.

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375144

  • 3.4. ELISA for human IL-877

    3.4.1. Selection of a partner antibody for N11 mAb in

    ELISA

    To develop a sandwich immunoassay specific for

    IL-877, we used a combination of N11 with a second

    mAb recognizing both IL-872 and IL-877. A panel of

    mAbs to IL-872 that were previously described (BS-1

    and WS-4, Ko et al., 1992) or produced in the current

    study (3A, 4C, H6 and N6), was tested to select the best

    partner for N11. Fig. 6 shows the titration of IL-877 in

    an ELISA using immobilized N11 (A) with different

    biotinylated anti-IL-8 mAbs for detection and the

    reverse configurations using biotinylated N11 (B).

    When N11 was used for capture, BS-1, H6 and N6

    were the best for detection, although WS-4 showed

    lower sensitivity. In the reverse configuration using

    biotinylated N11 for detection, the same mAbs showed

    reversed sensitivity with the WS-4/N11 combination

    being the most sensitive (Fig. 6B). Antibody 4C was

    unable to detect IL-877 when paired with N11 (data not

    shown).

    3.4.2. Comparison of ELISAs using the N11 mAb for

    either capture or detection

    Since both forms of IL-8 may be produced simulta-

    neously (Yoshimura et al., 1989; Schroder et al., 1990),

    an ELISA for IL-877 must be able to detect IL-877 in the

    presence of various concentrations of IL-872. We

    compared the efficiency of N11 for either capture or

    detection of IL-877 in the presence of increasing con-

    centrations of IL-872. Fig. 7A shows that the recog-

    nition of IL-877 was inhibited by the presence of

    increasing concentrations of IL-872, when mAb H6

    that recognizes both forms of IL-8 was used as the

    capturing antibody. In contrast, recognition of IL-877 in

    an ELISA using N11 to capture IL-877 was not affected

    in the presence of IL-872. The combination of N11 as

    the capturing antibody and biotinylated BS-1 or H6

    for detection was selected for further study. The detec-

    tion limit of this ELISA for IL-877 was between 25 and

    50 pg/ml (Fig. 7B).

    The ELISA using mAbs BS-1, WS-4 or 4C (rec-

    ognizing IL-872) for capture and biotinylated N6, H6

    or BS-1 for detection recognized both forms of IL-8

    and was called the total IL-8 ELISA. The total

    ELISA using BS-1 and N6 and the ELISA for IL-877showed similar IL-8 titration curves both in PBS0.3

    M NaCl0.5% BSATw and normal donor plasma

    (Fig. 7B and C). Since the total IL-8 determined by

    this assay is the sum of IL-872 and IL-877, the

    concentration of IL-872 in the samples was estimated

    by subtracting the concentration of IL-877 from the

    total IL-8.

    3.5. Production of IL-877 by cultured cells

    The ELISA for IL-877 was used to study the

    production of IL-877 in various cell cultures. We

    determined the concentration of IL-877 in supernatants

    from the cultures of endothelial cells, epithelial tumor

    cell lines, neuroblastoma cell lines SH and MC,

    myelo-monocytic cells THP-1, neutrophils and mono-

    cytes. Cells were cultured in media either with or

    Fig. 6. Comparison of ELISAs for IL-877 using different anti-IL-872mAbs paired with the anti-IL-877 mAb N11 used as the capture

    antibody (A) or as the detection antibody (B).

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 45

  • without serum and resting or stimulated with LPS,

    TNF-a or IL-1-h. Both forms of IL-8 were detected inthe supernatants from all cell cultures. The levels of

    IL-877 ranged from 5% to 70% of the total IL-8

    protein and were more dependent on the duration of

    cell culture and the presence of serum in the culture

    medium than on cell type (data not shown). To better

    understand the mechanisms of the generation of IL-

    877 in cell cultures, we studied the production of IL-

    877 and IL-872 by human monocytes. In monocytes

    stimulated with LPS for 3.5 h in the presence of the

    Fig. 8. The effect of protease inhibitors on the accumulation of IL-

    877 and total IL-8 in supernatants of LPS-stimulated monocytes. The

    plastic adherent fraction of human blood mononuclear leukocytes

    (adherence to 24-well plastic plates at 106/ml in 1 ml for 1 h) was

    stimulated with LPS (100 ng/ml) and incubated in the absence (A)

    or in the presence (B) of a protease inhibitor cocktail in 0.5 ml

    RPMI2% FCS at 37 jC for the indicated times. Supernatants werestored frozen at 40 jC after the addition of the protease inhibitorcocktail to all samples.

    Fig. 7. (A) Detection of IL-877 in the presence of IL-872 in an

    ELISA using mAb N11 as a capture antibody (E), compared to anELISA using mAb N11 as the detection antibody (D) paired withanti-IL-872 mAb H6. IL-877 was added to the capture antibody at 0.8

    ng/ml simultaneously with increasing concentrations of IL-872 (at

    0.62540 ng/ml). (B) Standard titration curves of IL-877 and IL-872in an ELISA using mAb N11 for capture and the biotinylated anti-

    IL-872 mAb H6 for detection. Open symbols: titration of IL-872 (5)and IL-877 (o) in PBS0.3 M NaCl0.5% BSA0.05% Tween-20; closed symbols: IL-872 (n) and IL-877 (.) were added tonormal donor plasma at 4 ng/ml and further diluted in PBS0.3 M

    NaCl0.5%BSA0.05%Tween-20. (C) The same titration of IL-872and IL-877 as in B using total ELISA consisting of anti-IL-872mAb

    BS-1 for capture and biotinylated mAb N6 for detection.

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375146

  • secretion inhibitor brefeldin, followed by lysis with

    Triton X-100 buffer containing protease inhibitors, no

    IL-872 was detected but levels of 100250 ng of IL-

    877 per 106 monocytes were found (data not shown).

    When the levels of both forms of IL-8 in the culture

    medium of monocytes stimulated with LPS were

    determined over the course of incubation, no IL-872was detected in the culture medium in the first 6 h

    (Fig. 8A). IL-872 was detected after 6 h with levels

    increasing with time with a corresponding decrease in

    levels of IL-877. By 12 h IL-877 comprised about 80%

    of the total IL-8. This decreased to less than 30% at 24

    h. When a cocktail of protease inhibitors was added at

    the beginning of incubation, only IL-877 was detected

    during the first 18 h (Fig. 8B). When cell-free super-

    natants from both 6 and 12 h monocyte cultures were

    incubated at 37 jC for 6 h, IL-877 was converted toIL-872. This could be prevented by adding a cocktail

    of protease inhibitors (data not shown). The conver-

    sion of the IL-877 to IL-872 was significant in serum

    containing media without any cells (about 75% of

    exogenous IL-877 was converted to the 72-amino-acid

    form during 20 h at 37 jC in RPMI10% FCS) andno conversion was detected in serum-free medium

    (data not shown).

    3.6. Detection of IL-877 in samples of plasma and

    cerebrospinal fluid

    Using the ELISA for IL-877 described above, we

    examined samples of human plasma and cerebrospinal

    fluid taken from patients with various forms of

    meningococcal infection and sepsis. To prevent pro-

    teolytic conversion of IL-877, blood and CSF were

    collected in plastic tubes containing both EDTA and a

    cocktail of protease inhibitors and were centrifuged

    not later than 1 h after blood collection to prevent

    possible IL-8 release from platelet or leukocyte depots

    (Su et al., 1996). The titration of IL-877 in normal

    donor plasma containing a PI cocktail was identical to

    that in PBS0.3 M NaCl0.5% BSATw (Fig. 7B).

    The content of IL-877 in 20 samples of normal

    volunteer donor blood was below the detection limit

    (data not shown); however, both IL-877 and IL-872were detected at various concentrations in the samples

    of plasma and CSF from patients with meningitis and

    sepsis (Table 2). It was surprising to find substantial

    levels of IL-877 in some samples of blood plasma that

    contained little or no total IL-8, as detected by a

    total IL-8 ELISA. This paradoxical combination

    was never observed in CSF samples from the same

    Table 2

    Detection of IL-877 and total IL-8 in human blood plasma and CSF from patients with meningitis and sepsis

    Patient Diagnosis Blood plasma CSF

    IL-877(ng/ml)

    Total IL-8 a

    (ng/ml)

    IL-877(ng/ml)

    Total IL-8

    (ng/ml)

    1 Meningitis of unidentified etiology 0.172 0.216 0.150 0.300

    2 Meningococcal meningitis, Meningococcemia 0.840 0.216 0.220 1.466

    3 Meningococcal meningitis, Meningococcemia < 0.060 < 0.015 1.808 7.375

    4 Purulent meningitis of unidentified etiology, fifth day 1.124 0.358 0.146 0.620

    4a Ibid, 11th day of disease 0.310 6.050 0.140 0.196

    5 Meningitis of unidentified etiology 1.562 < 0.015 n.t.b n.t.

    6 Hydrocephalia of unidentified etiology n.t. n.t. 3.804 5.544

    7 Meningococcemia 2.018 < 0.015 n.t. n.t.

    8 Meningococcemia 1.292 < 0.015 n.t. n.t.

    9 Meningoencephalitis 1.724 0.250 1.760 2.010

    10 Meningoencephalitis 0.361 0.604 16.150 42.150

    11 Encephalitis 2.036 0.225 0.654 2.250

    12c Major surgery, sepsis 1.432 0.132 n.t. n.t.

    a The data on total IL-8 were obtained using the BS-1-N6* ELISA; similar results were obtained using total IL-8 ELISAs 4C-H6*,

    WS-4-N6* and H6-4C* (* indicates biotinylated mAb).b n.t.non-tested.c Patient No.12 was an adult; all other patients were from the pediatric clinic, 9 months15 years old.

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 47

  • patients (Table 2), which showed elevated levels of

    IL-877 together with higher total IL-8.

    4. Discussion

    Because of the potential utility of measuring IL-877in culture supernatants, body fluids and tissue sec-

    tions, we produced a mAb specific for this form of IL-

    8 and developed a corresponding ELISA.

    To generate antibodies to the N-terminus of IL-877,

    mice were immunized with a recombinant antigen of

    IL-877 fused to the Fc portion of human IgG or with

    conjugates of the synthetic peptide AVLPRSA-

    KELRC-NH2, which corresponds to the N-terminal

    12 amino acids of IL-877.

    The use of FcIgGIL-877 in Freunds adjuvant for

    the priming and the first boost showed the immune

    response augmenting function of FcIgG, which may

    be relevant to the enhanced immunogenicity of fusion

    proteins or of antigens coupled to molecules that

    interact with immunocompetent cells such as C3b

    (Dempsey et al., 1996) and thyroglobulin (Mnich et

    al., 1995).

    However, the use of FcIgGIL-877 for the second

    boost showed no immunoenhancing effect and the

    antibody response to the Fc domain prevailed over

    the response to IL-8. While recombinant IL-8 was

    approximately three-fold weaker as an immunogen

    for priming and the first boost compared to FcIgG

    IL-877, it induced a three-fold higher antibody

    response to IL-8 when used for the second boost

    instead of FcIgGIL-877. We observed a similar loss

    of immunoenhancing function of a carrier protein

    after the second boost in the course of immunization

    against BSANTP77. The substitution of BSA for

    Ova as the carrier protein for the second boost

    markedly enhanced antibody responses to NTP77.

    This observation might be relevant to the so-called

    antigen competition phenomenon (Landsteiner,

    1945). It seems that the FcIgG fragment in recombi-

    nant FcIgGIL-877 and carrier proteins in conju-

    gates of NTP77 were necessary for efficient priming

    and the first boost, while they inhibited the antibody

    response toward the weaker antigens, IL-8 (Fig. 1)

    and NTP77, after the second boost. The N-terminus

    of IL-877 seems to be a weaker antigen compared to

    the FcIgG fusion partner, the 72-amino-acid body

    of IL-877 itself and even to the sulfo-MBS chemical

    bond between NTP77 and the carrier protein. Anti-

    IL-877 mAbs were produced by substituting both the

    carrier protein and the chemical bond in the NTP77conjugate in the course of immunization followed

    by a final pre-fusion immunization with synthetic

    IL-877.

    The mAb N11 that resulted from this protocol

    reacted with IL-877, FcIgGIL-877 and both the free

    and conjugated AVLPRSAKELRC-NH2 peptide of

    IL-877 but did not recognize IL-872. IL-877 may be

    converted to IL-872 by cleavage with serine proteases

    at the ArgUSer bond (Yoshimura et al., 1989; Hebertet al., 1990; Padrines et al., 1994) thus releasing the

    N-terminal AVLPR pentapeptide of IL-877. This pep-

    tide was shown to induce apoptosis in leukemic cells

    (Terui et al., 1999). N11 was unable to bind immobi-

    lized AVLPR peptide although the binding of N11

    with IL-877 and NTP77 was inhibited by the presence

    of an approximately 103 molar excess of AVLPR.

    These data indicate that the epitope recognized by

    N11 overlaps but does not coincide with the AVLPR

    sequence. In addition, these data demonstrate that the

    chance of inhibiting the recognition of IL-877 in an IL-

    877 ELISA by naturally occurring AVLPR peptide is

    rather small.

    The combination of N11 with H6 or BS-1 that

    recognize IL-872 was used to develop an IL-877-

    specific ELISA. It was shown that recognition of

    IL-877 was not affected by an excess of IL-872 added

    to the samples when N11, specific for IL-877, was

    used as the capture antibody. This ELISA showed

    specificity for IL-877 with a detection limit of 2550

    pg/ml, sufficient for measuring IL-877 in cell culture

    and human body fluids.

    The study of the production of IL-877 by mono-

    cytes and other cells in vitro has revealed that cultured

    cells secreted only the IL-877 form, while IL-872appeared at later time points of the cell culture due

    to post-secretion proteolytic degradation of IL-877 in

    the culture medium. This is in agreement with pre-

    vious observations using N-terminal sequencing and

    estimation of the molecular weight of IL-8 by SDS-

    PAGE (Yoshimura et al., 1989).

    To contact circulating blood leukocytes and induce

    their attachment and transendothelial emigration, IL-8

    must be exposed on the blood surface of endothelial

    cells (Rot, 1992; Tanaka et al., 1993; Rot et al., 1996;

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 375148

  • Middleton et al., 1997). The latest data indicate that

    IL-8 exposed on the circulation side of endothelium in

    response to LPS is produced by endothelial cells

    themselves, rather than in extravascular tissue

    (Dumont et al., 2000).

    Since it was shown here that IL-877 secreted by

    cultured cells persisted in vitro for a few hours and

    since leukocyte adherence and transendothelial emi-

    gration occur within a few minutes (Girard and

    Springer, 1995; Baggiolini, 1998), we suggest that

    the only form of IL-8 involved in neutrophilendo-

    thelium interactions in vivo is IL-877. The chance of

    fast intravascular proteolytic conversion of endothe-

    lium generated IL-877 to IL-872 is rather small since

    proteases in blood circulate with corresponding pro-

    tease inhibitors. We have detected no conversion of r-

    IL-877 to IL-872 after 20 h of incubation at 37 jC infresh normal donor plasma that was anticoagulated

    with EDTA and separated 30 min after venepuncture

    (data not shown). IL-877 was recently isolated from a

    wound healing area and was suggested to stimulate

    keratinocyte proliferation (Rennekampff et al., 2000).

    IL-872, on the other hand, is extremely resistant to

    further proteolytic degradation and might accumulate

    in inflammatory tissue reservoirs. In such places, IL-

    872 has been suggested to play a detrimental role in

    chronic inflammation (Yoshimura et al., 1989). The

    study of IL-877 and IL-872 localization in human

    tissue sections is currently performed using N11 and

    other mAbs. Since N11 is capable of neutralizing IL-

    877, it might be used in monkey models of inflamma-

    tion in vivo to explore the role of this form of IL-8 in

    leukocyte transendothelial emigration.

    To test which form of IL-8 is produced in vivo, we

    used samples of human plasma and cerebrospinal

    fluid taken from patients with sepsis and meningitis.

    IL-877 was clearly detected in these samples, indicat-

    ing that it is produced during inflammatory responses

    in vivo. It was surprising to find that high plasma

    levels of IL-877 in some of the blood samples were not

    accompanied by adequate levels of total IL-8 (i.e.

    the sum of the 72- and 77-amino-acid forms). These

    data imply that the total IL-8 ELISA was unable to

    detect IL-8 in some plasma samples, while the IL-877specific ELISA detected IL-877. This situation was

    never observed in the samples of CSF, where high

    levels of IL-877 were accompanied by the same or

    higher levels of total IL-8.

    Blockade of the recognition of IL-8 in plasma

    samples in a total IL-8 ELISA has long been

    known. Sylvester et al. (1992) have demonstrated

    that in human sera, IL-8 is bound with high affinity

    to IgG, which interferes with the ELISA and was

    suggested to serve as an IL-8 scavenger auto-

    antibody (Sylvester et al., 1992; Leonard, 1996;

    Kurdowska et al., 2001). We suggest that the total

    IL-8 ELISA in our experiments was blocked via the

    same mechanism. In favor of this suggestion is the

    absence of this situation in the samples of CSF that

    contain little or no IgG compared to plasma. Alpha

    2-macroglobulin has also been shown to form com-

    plexes with IL-8 (Kurdowska et al., 2000). Since the

    ELISA for IL-877 was not blocked in the same

    plasma samples, the N-terminal region of IL-877 is

    probably available for binding with N11 in hypo-

    thetical complexes. Interestingly, this phenomenon

    was observed in 21 plasma samples from 36 chil-

    dren, while in adult sepsis, it was found in 1 plasma

    from 12 patients. In a preliminary study using

    captured N11 and liquid phase antibody to human

    IgG (Sylvester et al., 1992), we detected IL-877/IgG

    complexes in the samples of human blood plasma.

    The investigation of this phenomenon is beyond the

    scope of this report.

    This observation addresses once more the often

    discussed problem: Immunoassays for detecting

    cytokines: What they are really measuring? (Mire-

    Sluis et al., 1995). While the use of an IL-877 ELISA

    as a diagnostic tool in diseases in which IL-8 may be

    implicated requires further investigation, the antibod-

    ies and assays described in this report should be useful

    in experimental studies of IL-8 in host defense and

    pathology.

    Acknowledgements

    We thank Dr. Bernard Scallon, Centocor Inc.,

    Malvern, PA, for the production of FcIgGIL-877 and

    the donation of FcIgGp-55; Prof. Marco Baggiolini,

    Kocher Inst., University Bern, Switzerland, for the

    gift of synthetic IL-877; Dr. Ernst Brandt, Forschungs-

    zentrum, Borstel, Germany, for providing the N-

    terminal peptide of human CXCR2; Anna Portyanko,

    Medical University, Minsk, Belarus, for help with

    immunohistology; Dr. Joost Oppenheim, LMI, NCI at

    N.N. Nashkevich et al. / Journal of Immunological Methods 270 (2002) 3751 49

  • Frederick, MD, for providing the BIOQUANT

    software for chemotaxis assays; Dr. Ilia Tichonov,

    Inst. Hematology, Minsk, Belarus, for help with

    mouse serum assays and production of mAb H6; Dr.

    Yuri Chaly, Inst. Hematology, Minsk, for his expert

    assistance in the manuscript preparation; Dr.

    Alexander Kudin, Medical University, Minsk, Bela-

    rus, for help in obtaining clinical samples of plasma

    and CSF; Dr. Ji Ming Wang, LMI, NCI at Frederick,

    MD, for the gift of recombinant human IL-877; and

    Prof. Sergei Ketlinsky, Institute of Highly Pure

    Biopreparations, St. Petersburg, Russia, for the gift

    of a rabbit antibody to human IL-8.

    The work was supported by Research Grant from

    Centocor Inc., Malvern, PA, a Grant from INTAS,

    Brussels, research funding from the Office of Interna-

    tional Affairs, Department of Health & Human

    Services, National Cancer Institute, NIH, Bethesda,

    MD, and from the Ministry of Health, Republic of

    Belarus.

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    IntroductionMaterials and methodsAnimals and reagentsConjugation protocolImmunizationMonoclonal antibodies: generation and purificationCharacterization of monoclonal antibodies to IL-8Hybridomas origin and isotyping dataChemotaxis assayImmunoblotsImmunohistochemistry

    ELISA detecting IL-8Induction of IL-8 in cell culturesDetection of IL-877 in clinical samples

    ResultsSerum antibody responses to FcIgG-IL-877 and conjugates of NTP77Specificity of monoclonal antibodies derived from mice immunized with FcIgG-IL-877 and NTP77 conjugatesCharacterization of antibody N11Specificity of mAb N11 in ELISACharacterization of N11 using immunoblotting and cell immunostainingN11 mAb is efficient in homologous ELISAAntibody N11 neutralizes chemotactic activity of IL-877

    ELISA for human IL-877Selection of a partner antibody for N11 mAb in ELISAComparison of ELISAs using the N11 mAb for either capture or detection

    Production of IL-877 by cultured cellsDetection of IL-877 in samples of plasma and cerebrospinal fluid

    DiscussionAcknowledgementsReferences

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