JOURNAL OF
`
`LEUKOCYTE
`
`BIOLOGY®
`
`Cellular and Molecular Mechanisms of Host Defense and Inflammation
`
`7TM Rec
`
`Ca 2.
`
`Rec
`
`Ras
`
`LC
`
`ATP (cid:9)
`
`cAMP
`
`PKA
`
`cAMP
`
`00
`
`cAMP
`
`Ras pathway
`
`target protein
`phosphorylation
`
`CYTOPLASM
`
`2.
`Ca
`
`target protein
`phosphorylation
`
`1
`
`PKC pathway
`
`Ins(1 4.5)P,
`
`2-
`
`AC cAMP / PKA
`pathway
`
`Ca'- / CaM pathway
`
`modulation of transactivating factors
`
`NUCLEUS
`
`regulation of cytokine
`gene transcription
`
`VOLUME 61, NUMBER 6, JUNE 1997
`Published by the Society for Leukocyte Biology ...N. 1r
`
`Ex. 1054 - Page 1
`
`

`

`JOURNAL OF
`
`LEUKOCYTE BIOLOGY
`
`An Official Publication of the Society for Leukocyte Biology
`
`Volume 61, Number 6 (cid:9)
`
`CONTENTS
`
`Reviews
`
`647 (cid:9)
`
`654 (cid:9)
`
`667 (cid:9)
`
`679 (cid:9)
`
`689 (cid:9)
`
`695 (cid:9)
`
`703 (cid:9)
`
`712 (cid:9)
`
`721
`
`729 (cid:9)
`
`736 (cid:9)
`
`745 (cid:9)
`
`753 (cid:9)
`
`759
`
`760
`
`767
`
`Mechanisms of neutrophil-induced parenchymal cell injury. H. Jaeschke and C. W. Smith
`
`lmmunosuppressive retroviral peptides. Immunopathological implications for immunosuppressive influ-
`ences of retroviral infections. S. Haraguchi, R. A. Good, G. J. Cianciolo, R. W. Engelman, and N. K. Day
`
`Pathophysiology
`
`Expression of TNF-a by human plasma cells in chronic inflammation. N. Di Girolamo, K. Visvanathan,
`A. Lloyd, and D. Wakefield
`
`Cell Development, Growth, Differentiation, and Function
`
`Monocytic-endothelial cell interaction: regulation of prostanoid synthesis in human coculture. S. Koll,
`M. Goppelt-Struebe, I. Hauser, and M. Goerig
`
`LPS-induced blood neutrophilia is inhibited by ai-adrenoceptor antagonists: a role for catecholamines. S. P.
`Altenburg, M. A. Martins, A. R. Silva, R. S. B. Cordeiro, and H. C. Castro-Faria-Neto
`
`Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils. J-M.
`Sallenave, M. Si-Ta har, G. Cox, M. Chignard, and J. Gauldie
`
`Complex regulation of human neutrophil activation by actin filaments: dihydrocytochalasin B and botuli-
`num C2 toxin uncover the existence of multiple cation entry pathways. K. Wenzel-Seifert, H. Lentzen,
`K. Aktories, and R. Seifert
`Timing of prostaglandin exposure is critical for the inhibition of LPS- or IFN-y-induced macrophage NO
`synthesis by PGE2. B. G. Harbrecht,Y-M. Kim, E. A. Wirant, R. L. Simmons, andT. R. Billiar
`
`Quantitation of surface CD14 on human monocytes and neutrophils. P. Antal-Szalmas, J. A. G. Van Strijp,
`A. J. L. Weersink, J. Verhoef, and K. P M. Van Kessell
`
`Extracellular Mediators and Effector Molecules
`
`IL-15 is chemotactic for natural killer cells and stimulates their adhesion to vascular endothelium. P Allavena,
`G. Giardina, G. Bianchi, and A. Mantovani
`
`Receptors, Signal Transduction, and Genes
`
`Primary structure of rat CD14 and characteristics of rat CD14, cytokine, and NO synthase mRNA expression
`in mononuclear phagocyte system cells in response to LPS. N. Takai, M. Kataoka, Y. Higuchi, K. Matsuura,
`and S.Yamamoto
`Inhititory effect of 3,4-dichloropropionaniline on cytokine production by macrophages is associated with
`LPS-mediated signal transduction. Y. C. Xie, R. Schafer, and J. B. Barnett
`Desensitization of the f MLP-induced NADPH-oxidase response in human neutrophils is lacking in okadaic
`acid-treated cells. 0. Harbecke, L. Liu, A. Karlsson, and C. Dahlgren
`
`Consulting Editors
`
`Subject Index to Volume 61
`
`Author Index to Volume 61
`
`COVER: Signal-transduction pathways via cell surface receptors. A negative or positive "cross talk" among these signal-transduction
`Pathways regulate cytokine gene transcription. Our hypothesis is that immunosuppressive retroviral sequences modulate type 1/type
`2 cytokine gene transcription by activating cAMP/PKA- and/or inhibiting PKC-dependent signaling pathways. See Haraguchi et al.,
`Pages 654-666.
`
`Ex. 1054 - Page 2
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`JOURNAL OF
`
`LEUKOCYTE BIOLOGY®
`
`An Official Publication of the Society for Leukocyte Biology
`
`Volume 61, Number 6
`
`CONTENTS
`
`June 1997
`
`647 (cid:9)
`654 (cid:9)
`
`667 (cid:9)
`
`679 (cid:9)
`
`689 (cid:9)
`
`695 (cid:9)
`
`703 (cid:9)
`
`712 (cid:9)
`
`721 (cid:9)
`
`729 (cid:9)
`
`736 (cid:9)
`
`745 (cid:9)
`
`753 (cid:9)
`
`759
`760
`767
`
`Reviews
`
`Mechanisms of neutrophil-induced parenchymal cell injury. H. Jaeschke and C.W. Smith
`Immunosuppressive retroviral peptides. Immunopathological implications for immunosuppressive influ-
`ences of retroviral infections. S. Haraguchi, R. A. Good, G. J. Cianciolo, R.W. Engelman, and N. K. Day
`
`Pathophysiology
`
`Expression of TNF-a by human plasma cells in chronic inflammation. N. Di Girolamo, K. Visvanathan,
`A. Lloyd, and D. Wakefield
`
`Cell Development, Growth, Differentiation, and Function
`
`Monocytic-endothelial cell interaction: regulation of prostanoid synthesis in human coculture. S. Koll,
`M. Goppelt-Struebe, I. Hauser, and M. Goerig
`LPS-induced blood neutrophilia is inhibited by aradrenoceptor antagonists: a role for catecholamines. S. R
`Altenburg, M. A. Martins, A. R. Silva, R. S. B. Cordeiro, and H. C. Castro-Faria-Neto
`Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils. J-M.
`Sallenave, M. Si-Ta har, G. Cox, M. Chignard, and J. Gauldie
`Complex regulation of human neutrophil activation by actin filaments: dihydrocytochalasin B and botuli-
`num C2 toxin uncover the existence of multiple cation entry pathways. K. Wenzel-Seifert, H. Lentzen,
`K. Aktories, and R. Seifert
`Timing of prostaglandin exposure is critical for the inhibition of LPS- or IFN-y-induced macrophage NO
`synthesis by PGE2. B. G. Harbrecht,Y-M. Kim, E. A. Wirant, R. L. Simmons, andT. R. Billiar
`Quantitation of surface CD14 on human monocytes and neutrophils. P. Antal-Szalmas, J. A. G. Van Strijp,
`A. J. L.Weersink, J. Verhoef, and K. P. M. Van Kessell
`
`Extracellular Mediators and Effector Molecules
`
`IL-15 is chemotactic for natural killer cells and stimulates their adhesion to vascular endothelium. P. Allavena,
`G. Giardina, G. Bianchi, and A. Mantovani
`
`Receptors, Signal Transduction, and Genes
`
`Primary structure of rat CD14 and characteristics of rat CD14, cytokine, and NO synthase mRNA expression
`in mononuclear phagocyte system cells in response to LPS. N. Takai, M. Kataoka, Y. Higuchi, K. Matsuura,
`and S.Yamamoto
`Inhititory effect of 3,4-dichloropropionaniline on cytokine production by macrophages is associated with
`LPS-mediated signal transduction. Y. C. Xie, R. Schafer, and J. B. Barnett
`Desensitization of the fMLP-induced NADPH-oxidase response in human neutrophils is lacking in okadaic
`acid-treated cells. 0. Harbecke, L Liu, A. Karlsson, and C. Dahlgren
`
`Consulting Editors
`Subject Index to Volume 61
`Author Index to Volume 61
`
`COVER: Signal-transduction pathways via cell surface receptors. A negative or positive "cross talk" among these signal-transduction
`pathways regulate cytokine gene transcription. Our hypothesis is that immunosuppressive retroviral sequences modulate type 1/type
`2 cytokine gene transcription by activating cAMP/PKA- and/or inhibiting PKC-dependent signaling pathways. See Haraguchi et al.,
`pages 654-666.
`
`Ex. 1054 - Page 3
`
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`JOURNAL OF
`
`LEUKOCYTE BIOLOGY®
`
`An Official Publication of the Society for Leukocyte Biology
`
`This journal will consider for publication manuscripts of original investigations
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`in host defense. These reports include full-length papers on original research,
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`
`EDITOR-IN-CHIEF, JOOST J. OPPENHEIM
`
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`Craig W. Reynolds
`
`Editorial Board
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`William S. Walker
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`
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`
`Vivian L. Braciale
`Hal E. Broxmeyer
`Myron Cybulski
`Charles J. Czuprynsky
`Howard E. Gendelman
`Thomas A. Hamilton
`Alan M. Kaplan
`Helen M. Korchak
`Margaret L. Kripke
`Alan L. Landay
`Kouji Matsushima
`
`Linda McPhail
`David M. Mosser
`Donna Paulnock
`Edgar Pick
`Ann Richmond
`Barrett J. Rollins
`Helene F. Rosenberg
`C. Wayne Smith
`Dennis E. Van Epps
`Sharon M. Wahl
`James R. Zucali
`
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`
`Ex. 1054 - Page 4
`
`

`

`Expression of TNF-a by human plasma cells in
`chronic inflammation
`Nick Di Girolamo, Kumar Visvanathan, Andrew Lloyd, and Denis Wakefield
`Inflammation Research Unit, School of Pathology, The University of New South Wales, Sydney, Australia
`
`Abstract: Tumor necrosis factor-a (TNF-a) is a potent
`pro-inflammatory cytokine and mediator of the inflam-
`matory response. It has been implicated in the patho-
`genesis of many inflammatory disorders, including
`rheumatoid arthritis (RA), septic shock, and Crohn's
`disease. Using a specific anti-human TNF-a antibody
`we detected immunoreactivity for this cytokine in the
`cytoplasm of inflammatory cells in several chronic
`inflammatory disorders, including RA, scleritis, and
`polyarteritis nodosa. These cells were identified pre-
`dominantly as IgG-expressing plasma cells. Lymph
`nodes from patients with Hodgkin's lymphoma and
`breast cancer, but not from control subjects, were also
`found to contain TNF-a-positive plasma cells. Cultured
`EBV-B lymphocytes and a human plasma cell line
`(ARH-77) when stimulated with phorbol myristate ace-
`tate demonstrated cytoplasmic TNF-a immunoreactiv-
`ity. Western blot analysis of cell membranes and condi-
`tioned media from both cell types revealed the presence
`of the 26-kDa membrane-bound form and the 17-kDa
`soluble form of TNF-a, respectively. TNF-a was quan-
`titated by enzyme-linked immunosorbent assay and
`found to be biologically active as determined by the
`L929 cytotoxicity assay. This is the first demonstration
`that plasma cells may be capable of modulating im-
`mune and inflammatory responses, not only by anti-
`body production, but also by their secretion of a key
`inflammatory mediator, TNF-a. J. Leukoc. Biol. 61:
`667-678; 1997.
`
`Key Words: B lymphocyte • cytokine • rheumatoid arthritis • scleritis
`
`synovial cells [11, 13], as well as activated monocytes and
`macrophages [14]. The TNF-a gene encodes a surface
`transmembrane, biologically active, 26-kDa precursor pro-
`tein that can act locally through cell-to-cell contact. Process-
`ing of this cell-bound protein appears to arise from proteo-
`lytic cleavage by a member(s) of the matrix metalloproteinase
`(MMP) gene family [15-17], resulting in shedding of an
`active 17-kDa soluble protein [18]. Specific functions of
`TNF-a include the following: mediation of prostaglandin
`and interleukin-1 (IL-1) synthesis [19], induction of adhe-
`sion molecule expression [20, 21], induction of connective
`tissue-degrading enzymes of the MMP class [22, 23], and
`B cell activation [24].
`Although TNF-a expression and synthesis has been ex-
`tensively investigated in culture models, few studies have
`sought to localize and characterize cells expressing this cy-
`tokine in vivo in chronic inflammatory diseases such as
`R.A. Despite the detection of increased levels of TNF-a in
`synovial fluid from patients with RA [4], it is not entirely
`clear whether this cytokine is produced locally within the
`joint. Mononuclear cells isolated from RA synovial joints
`have been shown to express elevated amounts of TNF-a as
`well as other cytokines, both at the mRNA and protein levels
`[13]. Other studies using immunohistochemical techniques
`have localized TNF-a protein to endothelial and macro-
`phage type-A synoviocytes in RA tissue sections [11, 14].
`In this investigation, surgical tissue specimens were ob-
`tained from patients with various vasculitic and chronic
`inflammatory disorders to elucidate the potential role of
`TNF-a at the site of disease activity and to reveal the cel-
`lular source(s) of this multifunctional cytokine.
`
`ti
`
`INTRODUCTION
`
`Tumor necrosis factor a (TNF-a) is a pleiotropic cytokine
`[1] that plays a key role in inflammation. Originally de-
`scribed for its anti-tumor activity [2], this pro-inflammatory
`cytokine is implicated in the pathology of rheumatoid arthri-
`tis (RA) [3, 4], septic shock [5], cerebral malaria [6], and
`cancer [7, 8]. Secretion of this cytokine can be induced in
`vitro by phorbol esters such as phorbol myristate acetate
`(PMA) or lipopolysaccharide (LPS). TNF-a is produced by
`a variety of cell types including the following: T lympho-
`cytes [9, 10], endothelial cells [11], B lymphocytes [9, 12],
`
`Abbreviations: TNF-a, tumor necrosis factor a; RA, rheumatoid ar-
`thritis; PAN, polyarteritis nodosa; PMA, phorbol myristate acetate; IHC,
`immunohistochemistry; LPS, lipopolysaccharide; MMP, matrix metallo-
`proteinase; TES, 3-aminopropyltriethoxy-silane; TBS, Tris-buffered sa-
`line; DAB, 3,3'-diaminobenzidine tetrahydrochloride; rhTNF-a, recom-
`binant human TNF-a; FCS, fetal calf serum; ELISA, enzyme-linked
`immunosorbent assay; PBS, phosphate-buffered saline; SFM, serum-
`free media; CM, conditioned medium.
`Correspondence: Professor Denis Wakefield, Inflammation Research
`Unit, School of Pathology, The University of NSW, Sydney 2052, Aus-
`tralia.
`Received November 28, 1996; revised March 6, 1997; accepted
`March 7, 1997.
`
`Journal of Leukocyte Biology Volume 61, June 1997 667
`
`Ex. 1054 - Page 5
`
`

`

`Immunoreactivity for TNF-a in Tissue Sections From
`TABLE 1. (cid:9)
`Patients with Chronic Inflammatory Diseases (1-11) and
`Non-Inflammatory Disorders (23 and 24)
`
`Diagnosis
`
`RA
`RA
`
`PAN
`PAN
`Scleritis
`
`Tissue
`Cyst
`Synovium
`
`Lung
`Skin
`Sclera
`
`Specimen (cid:9)
`1
`2, 3
`
`4
`5
`6-9
`
`10
`
`11
`12, 13
`14, 15
`16, 17
`18-22
`23
`24
`
`TNF localized to
`Plasma cells
`Plasma cells
`Synovial lining
`Fibroblasts
`Plasma cells
`Fibroblasts
`Plasma cells
`Fibroblasts
`Endothelial cells
`Plasma cells
`Fibroblasts
`Plasma cells
`Lung
`Tuberculosis
`No cells
`Sclera
`Normal
`No cells
`Synovium
`Normal
`No cells
`Skin
`Normal
`Lymph node No cells
`Normal'
`Plasma cells
`Lymph node
`Breast cancer
`Plasma cells
`Hodgkin's lymphoma Lymph node
`Reed-Sternberg
`Endothelial cells
`
`Empyema
`
`Pleura
`
`a Lymph nodes derived from patients undergoing carotid endarterectomy.
`
`MATERIALS AND METHODS
`
`Diseased and normal tissue specimens
`
`Stored tissue specimens were obtained from archival material in the De-
`partment of Anatomical Pathology, The Prince of Wales Hospital, and
`Department of Eye Pathology, Sydney Eye Hospital, Sydney, Australia.
`The clinical diagnoses and sources of patient tissue are summarized in
`Table 1.
`
`Immunohistochemical analysis
`
`Two-micrometer serial, control, and diseased tissue sections were cut
`from formalin-fixed, paraffin-embedded blocks, mounted on 3-amino-
`propyltriethoxy-silane (TES)-coated slides, and dried at 37°C. Tissue
`was de-paraffinized in Histoclear (Medos, Sydney, Australia) and dehy-
`drated through a graded series of ethanols and processed for immuno-
`histochemistry (IHC). Sections were incubated in 0.8% pepsin (Dako
`Corp., Carpinteria, CA), in 0.01 N HC1 at 37°C for 20 min or in 10
`Ag/mL proteinase K (Boehringer Mannheim, Sydney, Australia) at 37°C
`for 20 min, followed by two 5-min washes in 0.05 M Tris-buffered saline
`(TBS; 10x stock: 0.25 M Tris base, 0.25 M Tris-HC1, 8.5% NaC1, pH
`7.6). Endogenous peroxidase was quenched with 3% hydrogen peroxide/
`methanol for 5 min, then washed in TBS and the sections incubated with
`a 1:5 dilution of horse serum for 20 min. Mouse anti-human IgG, anti-
`human CD20, anti-human factor VIII-related antigen (Dako), and mouse
`anti-human TNF-a antibody (Genzyme Diagnostics, Cambridge, MA) were
`used as primary antibodies at dilutions of 1:50, 1:30, 1:50, and 1:100,
`respectively. The anti-human TNF-a monoclonal antibody does not cross-
`react with TNF-11 (Genzyme Diagnostics). Sections were incubated for
`25 min, after which a 1:200 dilution of biotinylated horse anti-mouse
`secondary antibody (Vector Laboratories, Burlingame, CA) was applied
`for 20 min. Avidin-biotin-peroxidase complex was then added (Vector
`Laboratories) for 30 min, and the color developed with 0.03% 3,3'-
`diaminobenzidine tetrahydrochloride (DAB; Sigma, Sydney, Australia)
`in TBS containing 0.006% H202. Sections were lightly counterstained
`with hematoxylin. Specificity of the reaction was verified by omitting the
`primary antibody and by pm-absorbing the anti-TNF-a antibody with
`recombinant human TNF-a (rhTNF-a; R & D Systems, Minneapolis, MN)
`
`668 Journal of Leukocyte Biology Volume 61, June 1997
`
`overnight at 4°C, then applying this solution to the tissue section. Ad-
`ditional controls included incubating sections in pre-immune mouse se-
`rum in place of the primary antibody. Control tissue included normal
`lymph nodes from healthy subjects, lymph node from a patient with breast
`cancer, a lymph node from a patient with Hodgkin's lymphoma (known
`to contain plasma cells), and normal scleral, synovial, and skin tissue.
`Sections were viewed under the light microscope (Zeiss, Oberkochen,
`Germany) and photographs taken using Kodak Ektachrome EPP-100 film.
`No attempt was made to grade the signal intensity for TNF-a between
`tissue samples because the specimens were not collected or fixed at the
`same time with a standardized protocol.
`
`EBV-B lymphocytes and plasma cell (ARH-77) cultures
`Normal human B lymphocytes were transformed with EBV as previously
`described [25]. Previous immunohistochemical analyses on this cell line
`revealed abundant CD20 surface antigen expression, with little or no
`IgG immunostaining (unpublished observations). The ARH-77 plasma
`cell leukemia line (expressing IgGi x-light chains; American Type Cul-
`ture Collection, Rockville, MD) was cultured in 75-cm2 tissue culture
`flasks (Nunc, Roskilde, Denmark) containing RPMI 1640 (Trace Biosci-
`ences, Sydney, Australia) with 10% fetal calf serum (FCS; CSL, Melbourne,
`Australia) and 100 units/mL penicillin and 100 Ltg/mL streptomycin
`(Trace Biosciences, Sydney, Australia). Similar immunohistochemical
`analyses on this plasma cell line revealed cytoplasmic staining for IgG,
`with no expression of surface CD20 (data not shown). Passaged cells
`were split 1:3 twice a week. All cell culture media and solutions used
`were filtered through Zeta-pore filters (Cuno Filter Systems, Sydney,
`Australia) to remove any contaminating endotoxin. Endotoxin levels were
`monitored in the cultures with the use of a Limulus amebocyte lysate
`assay (Associates of Cape Cod, Falmouth, MA). For enzyme-linked im-
`munosorbent assay (ELISA) and Western blotting assays, cells were
`seeded into 75-cm2 culture flasks and grown in 10% FCS/RPMI until
`enough cells were propagated for experimental use. Cells were centri-
`fuged at 1700 rpm, culture media removed, before washing three times
`with phosphate-buffered saline (PBS) and three times with RPMI. The
`cells were left in serum-free media (SFM; 0.2% BSA/RPMI) for 24 h,
`after which the SFM was removed. The cells were counted and plated
`in fresh SFM with 0-1000 ng/mL PMA (Sigma). Conditioned medium
`(CM) was harvested and stored in small aliquots at — 70°C until it was
`used for biochemical analyses.
`
`Cell membrane extraction
`
`EBV-B lymphocytes and human plasma cells were cultured as above un-
`der SF conditions, and stimulated with or without PMA. Cells were
`washed three times in PBS, then resuspended and allowed to swell for
`10 min on ice in buffer containing 0.003 mM NaC1, 3 mM Tris-Ha,
`pH 8.1, and 0.005 M MgC12 with 1 mM (final) phenylmethylsulfonyl
`fluoride (Sigma) and 50 1.tg/mL soybean trypsin inhibitor (Sigma). The
`preparations were homogenized and centrifuged at 10,000 rpm for 20
`min. The resulting pellet was resuspended in a Nonidet P-40 (NP-40)
`buffer containing 50 mM Tris-HC1, pH 7.4, 0.5 M NaC1, 5 mM ethylene-
`diaminetetraacetate, 0.5% NP-40, and the inhibitors used above at the
`same concentration. Finally, the suspension was centrifuged at 10,000
`rpm for 20 min and the supernatants containing NP-40 extracts col-
`lected and stored at —70°C until analysis.
`
`Western blot analysis
`
`CM and membrane extracts were electrophoretically separated on a 4%
`stacking, and a 10% resolving, acrylamide gel under non-reducing condi-
`tions. Proteins were transferred to nitrocellulose membranes (Schleicher
`& Scheull, Dassel, Germany) using a Trans-Blot Semi-Dry Electropho-
`retie Transfer Cell (Bio-Rad, Sydney, Australia) for Western blot analysis
`as previously described [26]. After transfer, the membranes were briefly
`washed in TBS containing 0.1% Tween-20 (TBST), then blocked in
`TBST containing 3% skim milk powder and 3% BSA for 1 h at room
`temperature. The membranes were again washed briefly in TBST, then
`incubated for 1 h at room temperature with gentle shaking in a 1:500
`
`Ex. 1054 - Page 6
`
`

`

`dilution of a biotinylated polyclonal rabbit anti-human TNF-a antibody
`(Genzyme Diagnostics) in 2% BSA/TBST. Membranes were then washed
`four times in TBST, for 15 min each, and a 1:1000 dilution of horse-
`radish peroxidase-conjugated Streptavidin (Dako) in 2% BSA/TBST in-
`cubated with membranes for 1 h shaking. Membranes were washed five
`times for 15 min each in TBST. A chemiluminescent reagent for non-
`radioactive detection of proteins was added (DuPont, Sydney, Australia)
`for 1 mM shaking. Excess reagent was removed by blotting the mem-
`branes on Whatman 3M paper; the immune complexes were visualized
`by exposing the membranes to Kodak X-OMAT AR scientific imaging
`film.
`
`ELISA
`
`CM were thawed and analyzed by commercial ELISA (DuoSet) to deter-
`mine TNF-a secretion by cultured plasma cells and B lymphocytes. As-
`say conditions were as per manufacturer's specifications (Genzyme Di-
`agnostics). Briefly, the assay required a capture antibody (specific for
`only TNF-a) that coated the wells of a microtiter plate. TNF-a standards
`and CM samples were applied in triplicate and incubated for 1 h at
`37°C on antibody-coated plates. The plates were washed three times
`and a biotinylated rabbit anti-human TNF-a antibody (identical to that
`used for Western blotting) was added to each well and incubated for
`1 h at 37°C. Plates were then washed and incubated with horseradish
`peroxidase-conjugated streptavidin (Genzyme kit). The microtiter plates
`were washed extensively, ABTS substrate added, and the absorbance
`read at 405 nm on a Titertek Multiscan Plus MKII plate reader (ICN-
`Flow, Sydney, Australia). The amount of TNF-a present in CM samples
`was calculated from the standard curve.
`
`L929 cytotoxicity assay
`
`The murine fibroblast cell line L929 was maintained in 10% FCS/
`RPMI, 2 mM L-glutamine, 100 units/mL penicillin, and 100 gg/mL
`streptomycin. Cells were counted and seeded at 3 x 105 cells/mL into
`the wells of a 96-well flat-bottomed cell culture plate (Costar Corp., Cam-
`bridge, MA), incubated for 24 h, and used as targets in this assay as
`previously described [24 Supernatants were removed and replaced
`with 50 4/well of 1 .tg/mL actinomycin D (Sigma) and 100 1.1L/well
`of CM samples, tested in triplicate. After an 18-h incubation at 37°C,
`the supernatants were removed and the viable fibroblasts fixed and
`stained for 5 mM with 100 4/well of a solution containing 0.5% crystal
`violet, 8% formaldehyde, 0.17% NaCl, and 22.3% ethanol made in ster-
`ile distilled water. The plates were washed extensively and rigorously
`with water and allowed to air dry. A solution containing 33% acetic acid
`and 1% Triton X-100 in water (100 µL/well) was used to dissolve the
`cell-bound crystal violet. The absorbance was measured at 540 nm
`using the same plate reader as above. The amount of bioactive TNF was
`determined from the standard curve generated.
`
`Statistical methods
`
`Statistics were performed with the use of unpaired Student's t-tests to
`compare results from each cell line after PMA stimulation, with results
`from untreated control cells. Data are expressed as mean ± Si). Results
`were considered statistically significant when P < 0.05.
`
`RESULTS
`
`Immunoreactivity for TNF-a in various
`Inflammatory diseases
`Tissue was obtained from 11 patients with chronic inflam-
`matory diseases: RA (n = 3), scleritis (n = 4), polyarteritis
`nodosa (PAN; n = 2), empyema (n = 1), and active pul-
`monary tuberculosis (n = 1). A common feature of the dis-
`
`eased tissues studied included the infiltration of inflamma-
`tory cells. Among these inflammatory cells were plasma
`cells, characterized morphologically by their distinctive
`clock-faced nuclei. These cells were generally localized to
`the periphery of inflammatory lesions or granulomas, par-
`ticularly in necrotizing scleritis. Immunohistochemically,
`the cells were identified as plasma cells using an anti-
`human IgG antibody, with prominent cytoplasmic immuno-
`reactivity for this antigen (Fig. 1A and F).
`IgG-positive plasma cells expressing cytoplasmic TNF-a
`were abundant in several diseases studied, including necro-
`tizing scleritis and RA (Fig. 1). Other cells that displayed
`immunoreactivity for TNF-a in necrotizing scleritis included
`resident scleral fibroblasts, large macrophage-like cells,
`and factor VIII-positive endothelial cells (Fig. 1D). A sim-
`ilar pattern of immunoreactivity for TNF-a was observed
`in RA tissue. In addition to revealing moderate TNF-a re-
`activity in synovial lining macrophage type-A synoviocytes
`(Fig. 1H), slightly weaker staining of the fibroblastic type-B
`synoviocytes of the deeper connective tissue was noted in
`all RA synovial tissue studied (micrographs not shown).
`Similar results were obtained in other disease tissue. In ad-
`dition, examination of normal scleral, synovial, and skin tis-
`sue by this technique resulted in no detectable IgG or TNF-
`a staining (micrographs not shown). There was no TNF-a
`immunoreactivity in control sections incubated with anti TNF
`antibody pre-absorbed with rhTNF-a (Fig. 1, C and G).
`Omitting the primary antill'NF-a antibody resulted in no
`staining (micrographs not shown). Table 1 summarizes the
`tissue samples examined and the characterized cells in which
`TNF-a was localized. There were no CD20-positive B lym-
`phocytes expressing TNF-a in any of the sections examined.
`
`Immunoreactivity for TNF-a in normal and
`diseased lymph nodes
`To determine whether plasma cells expressed TNF-a in
`non-inflammatory disorders, 2-pm serial tissue sections
`were cut from normal or malignant lymph nodes and pro-
`cessed for IHC. Tissue sections from Hodgkin's lymphoma
`lymph nodes contained many IgG-positive plasma cells,
`specifically located within the fibrous connective tissue net-
`work (Fig. 2C, arrows). These cells demonstrated signifi-
`cant immunoreactive staining for TNF-a (Fig. 2B, arrows).
`Although the staining was specific to plasma cells, it was
`by no means exclusive, with endothelial cells and Reed-
`Sternberg cells also expressing TNF-a (micrographs not
`shown). In addition, this node was heavily infiltrated by
`CD20-positive B lymphocytes (Fig. 2A, arrowheads), which
`were consistently negative for TNF-a (Fig. 2B, arrowheads).
`Although IgG-positive plasma cells were observed in quies-
`cent normal lymph nodes (Fig. 2E), little or no TNF-a
`expression was detected (Fig. 2F), whereas the lymph node
`infiltrated with tumor cells and containing similar numbers
`of IgG-positive plasma cells expressed TNF-a (micrographs
`not shown). Specificity of the TNF-a immunoreaction was
`determined as illustrated in Figure 1 by deleting the pri-
`mary antibody (micrograph not shown) and by pre-absorbing
`
`Di Gitolamo et al. Production of TNF-a by human plasma cells 669
`
`1
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`Ex. 1054 - Page 7
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`670 Journal of Leukocyte Biology Volume 61, June 1997
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`Ex. 1054 - Page 8
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`the anti-TNF-a antibody with rhTNF-a (Fig. 2D). This data
`is also summarized in Table 1.
`
`lmmunoreactivity for TNF-a in cultured human
`plasma cells
`
`Having demonstrated the abundance of TNF-a expression
`in plasma cells in vivo we sought to determine whether TNF-
`a could be detected in cultured human plasma cells. A
`plasma cell leukemia (ARH-77) cell line was cultured un-
`der normal SF, endotoxin-minimized conditions to deter-
`mine whether these cells, like their in vivo counterparts,
`expressed TNF-a (Fig. 3, A—C). In parallel, EBV-B lym-
`phocytes were cultured under identical conditions (Fig. 3,
`D—F), the cells were pelleted, formalin-fixed, and pro-
`cessed for paraffin sectioning (Materials and Methods). Se-
`rial, 2-1.1,m cell pellet sections were used for IHC. TNF-a
`was immunolocalized specifically to the cytoplasm of plas-
`ma cells (Fig. 3, A and B) and was consistent with the re-
`sults obtained in diseased tissue sections (Figs. 1 and 2). An
`identical immunostaining pattern for TNF-a was observed
`in cultured EBV-B lymphocytes (Fig. 3, D and E). How-
`ever, exposure to PMA resulted in a dramatic loss of TNF-a
`immunoreactivity, down from approximately 70-80% TNF-
`a-positive cells in control cultures (Fig. 3, A and D) to
`10-20% TNF-a-immunoreactive cells after PMA stimula-
`tion (Fig. 3, B and E). In addition, the intensity of immuno-
`staining was decreased after PMA stimulation, suggesting
`a down-regulation of TNF-a expression or an increase in
`the shedding of this cytokine. The specificity of the reac-
`tion was determined as per Figures 1 and 2, and included
`the deletion of the primary anti-TNF-a antibody step (Fig. 3,
`C and F), and antiTNF-a antibody pre-absorption with
`rhTNF-a (micrographs not shown).
`
`Western blot analysis for TNF-a on membrane
`extracts and CM
`
`Additional protein detection assays were employed to cor-
`roborate the immunohistochemical data. Membrane ex-
`tracts were prepared from cultured B lymphocytes and
`plasma cells for analysis by Western blotting. Using a poly-
`clonal anti-human TNF-a antibody, a specific immunopre-
`cipitant band was revealed at approximately 26-kDa, corre-
`sponding to the membrane-bound form of TNF-a (Fig. 4,
`top panel). This immunoreactive band was induced by stim-
`ulating