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Characterization of Human Fibroleukin, a Fibrinogen-Like Protein Secreted by T Lymphocytes¹

Susanna Marazzi, Stephanie Blum^*, Reto Hartmann^*, Doris Gundersen^*, Magali Schreyer, Scott Argraves, Vladimir von Fliedner, Robert Pytela^§ and Curzio Rüegg^2,*

^* Centre Pluridisciplinaire d’Oncologie, School of Medicine, University of Lausanne, Lausanne, Switzerland; Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland; Department of Cell Biology, Medical University of South Carolina, Charleston, SC 29425; and ^§ Lung Biology Center, Department of Medicine, University of California, San Francisco, CA 94143

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the ß- and -chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4⁺ and CD8⁺ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3⁺/CD45R0⁺) compared with naive T lymphocytes (CD3⁺/CD45RA⁺). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interactions of T lymphocytes with extracellular matrices or cell surfaces are important for T lymphocyte activation and function. For example, target cell killing, helper activity, extravasation, and migration into inflammatory sites are all dependent on T lymphocyte adhesion events. These interactions are mediated by a variety of adhesion receptors including integrins, Ig superfamily members, selectins, and CD44 (for review, see Refs. 1 and 2).

There is substantial in vitro evidence indicating that interactions with extracellular matrix (ECM)³ components not only play a role in adhesion and migration, but also affect T lymphocyte activation and effector functions. For example, adhesion to immobilized fibronectin or laminin during T lymphocyte stimulation results in an enhanced proliferative response (3, 4, 5). Adhesion of T lymphocytes to fibronectin or laminin in the presence of monocytes has been shown to induce TNF secretion (6). These in vitro findings suggest that T lymphocyte-ECM interactions in vivo may not only mediate adhesion and migration at tissue sites but that they may also participate in the modulation of local T lymphocyte function (reviewed in Refs. 7–9). For example, it has been suggested that in rheumatoid arthritis, deposition of fibronectin in the inflamed synovium may play a role in both the accumulation of T lymphocytes in the synovium and in providing costimulatory signals for T lymphocyte activation (10, 11, 12). Further evidence supporting a central role of ECM proteins in T lymphocyte function is suggested by the observation that T lymphocytes can produce matrix proteins. Activated T lymphocytes secrete fibronectin, which strongly supports lymphocyte adhesion and acts as a potent monocyte agglutinating factor (13, 14). The matrix protein ETA-1/osteopontin is secreted early during T lymphocyte activation and causes recruitment of monocytes/macrophages in vivo (15). Its expression is associated with an increased resistance of mice to infection with the intracellular bacterium Rickettsia tsutsugamushi (16). ETA-1/osteopontin was recently shown to induce cellular chemotaxis in vitro by binding to the receptor molecule CD44 (17). Taken together, these findings suggest that T lymphocyte-derived ECM and adhesion-related proteins may play a cytokine-like role in T lymphocyte physiology. According to this hypothesis, one may predict the identification of additional matrix/adhesion-related proteins produced by T lymphocytes, the functional role of which may be more specific to the immune system compared with the ubiquitous proteins fibronectin and osteopontin. A cDNA, termed pT49, encoding a candidate molecule with such potential characteristics has been identified by Koyama and coworkers using subtractive T minus B lymphocyte cDNA library screening (18).

We have previously reported the cloning and sequencing of the human pT49 homologue (hpT49h) cDNA (19). The deduced sequences of the human and murine proteins are 80% identical. We found mRNA expression in human peripheral blood T lymphocytes, in a B lymphocyte-derived line, but not in a variety of lymphoid and nonlymphoid cell lines (19). The putative protein encoded by the pT49 gene shows a strong homology to fibrinogen ß- and -chains. Its carboxyl-terminal half consists of a so-called fibrinogen-related domain (FRED), that is homologous to domains present on other extracellular proteins, while the amino-terminal half is predicted to contain heptad repeats and Cys pairs similar to those observed in the fibrinogen -, ß-, and -chains. In the fibrinogen molecule, these elements allow the formation of stable coiled-coil interactions between the three chains, which are essential for the generation of the mature fibrinogen complex.

Here, we report the identification and the initial characterization of the hpT49h gene product, which we have termed fibroleukin to indicate its relationship to the extracellular matrix and leukocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Proteins and Abs

BSA, plasma fibronectin, and plasma fibrinogen were purchased from Sigma Chemicals, St. Louis, MO. Human tenascin C was obtained from Life Technologies, Basel, Switzerland. The anti-FLAG mAb M2 (IgG1) was purchased from Kodak-IBI (Integra Biosciences, Wallisellen, Switzerland). The rabbit Ig against human fibrinogen, rabbit anti-mouse Ig, peroxidase-conjugated goat anti-mouse Ig, donkey anti-rabbit Ig, and phycoerythrin (PE)-conjugated goat anti-mouse Ig were from Caltag (South San Francisco, CA) and Dako (Zug, Switzerland). The horse anti-mouse biotinylated Ig and the avidin-peroxidase conjugate were from Vector Laboratories (Burlingame, CA). Biotinylated and FITC-labeled mAbs to either CD3 (anti-Leu-4), CD4 (anti-Leu-3a), CD8 (anti-Leu-2a), FITC-labeled mAb against CD16 (anti-Leu-11a), CD19 (anti-Leu-12), CD45R0 (anti-Leu-45 R0), and CD45RA (anti-Leu-18) were from Becton Dickinson (San Jose, CA). Streptavidin-conjugated magnetic microbeads were from Myltenyi Biotech, Bergisch Gladbach, Germany. The mAb TR66 directed against the CD3 complex was a gift from Dr. S. Abrignani, Basel, Switzerland. Human rIL-2 was kindly provided by Dr. M. Nabholz, Epalinges, Switzerland. IFN- (3 x 10⁷ U/mg) was a gift from Dr. G. Adolf, Bender, Vienna, Austria.

Cell lines and culture conditions

The myeloma line Sp2/0 and the hepatocarcinoma line HepG2 were obtained from American Type Culture Collection, Manassas, VA. The human T cell line Jurkat was obtained from the Cell Culture Facility at the University of California, San Francisco (UCSF). The EBV-transformed human B cell line SLA was obtained from Dr. T. Springer, Center for Blood Research, Boston, MA. The human embryonic 293T line was obtained from Dr. J. Tschopp, Epalinges, Switzerland. Sp2/0 and hybridoma lines were cultured in RPMI 1640, while HepG2 and 293T cells were cultured in DMEM (UCSF Cell Culture Facility and Life Technologies, Basel, Switzerland), supplemented with 10% FCS (UCSF Cell Culture Facility, and Sera-Tech, St. Salvator, Germany), 100 U/ml-100 µg/ml penicillin-streptomycin and 2 mM L-Glu (UCSF Cell Culture Facility; and Seromed, Fakola, Basel, Switzerland) at 37°C, 5% CO₂, and 95% humidity. PBS was from UCSF Cell Culture Facility and Seromed.

Expression and purification of recombinant bacterial proteins

The cDNA coding the FRED of fibroleukin was amplified from the gt11 clone 86 (19) using the forward primer hpT49hF + 663 (5'-CCTTCCCGGGAAAGATTGCTCTGACTACTACGC-3') (for clone 129) and the reverse primers hpT49Rwt (5'-CCTTTCTAGATTATTATGGCTTAAAGTGCTTGGGTCTG-3'). Amplification was performed for 20 cycles (2 min at 96°C, 2 min at 43°C, and 5 min at 72°C) with Pfu polymerase (Stratagene, La Jolla, CA). After an additional incubation for 5 min at 72°C in the presence of Taq polymerase (Life Technologies), the product was cloned into pCR II plasmid (Invitrogen, Carlsbad, CA). After confirmatory sequencing (Sequenase 2.0 kit, U.S. Biochemical, Cleveland, OH, and [³⁵S]dATP, NEN-DuPont, Regensdorf, Switzerland), the products were subcloned into the vector pTrxFus (Invitrogen) in frame with the thioredoxin gene (clone 129) or into a vector (pGB1) where the coding sequence for thioredoxin was replaced by encoding the start Met and six His residues (clone 214). The calculated molecular mass of the Trx-FRED and 6His-FRED proteins is 42 and 25 kDa, respectively. The assembled plasmids and the respective control plasmids were introduced into GI174 cells (Invitrogen) by chemical transformation. For protein expression, clones were grown at 30°C to OD 0.5, then induced with L-Trp and further incubated for 4 to 5 h. Cells were recovered by centrifugation and lysed at 4°C in 6 M guanidinium thiocyanate. Insoluble material was removed by centrifugation, and the supernatant was dialyzed slowly, once against 4 M urea, 50 mM Tris, 300 mM NaCl, and once against 20 mM urea, 50 mM Tris, 300 mM NaCl (SpectraPor, Spectrum Medical Industries, Houston, TX). The Trx/Trx-FRED and the 6His-FRED proteins were isolated by affinity chromatography on Thiobond resin (Invitrogen) and Ni-NTA Superflow resin (Qiagen, Basel, Switzerland) following the manufacturers’ protocols. The eluted material was dialyzed against PBS and analyzed by SDS-PAGE (20).

Transient expression of recombinant proteins in 293T cells

For transfection experiments, the hpT49h cDNA encoding the putative mature protein was amplified by PCR from PBMC cDNA using the forward primer hupT49hF + 16 (5'-CTTGAATTCGGATCCACTTACGGTTTTTTGGTTGTGGC-3') and either of the reverse primers hupT49hRwt (see above) or hupT49hRFlag (5'-TTCCTTCCTTCTCGAGTTATTACTTGTCATCGTCGTCCTTGTAGTCTGGCTTAAAGTGCTTGGGTCT-3') at 60°C annealing temperature. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland, sequencing facility), and subcloned into the pCR3-based (Invitrogen) mammalian expression vector pS188 (provided by Dr. P. Schneider, Epalinges, Switzerland) in frame with the leader sequence of the Ig heavy chain gene. This sequence was used to replace the natural sequence coding for the predicted leader peptide of the hpT49h gene product to assure optimal secretion. Plasmid DNA for transfection was prepared using the Qiaprep system (Qiagen). Transfection of subconfluent 293T cells was performed with the calcium phosphate DNA precipitation method (21). At 12 h posttransfection, culture medium was removed and cells further cultured in OptiMEM (Life Technologies) in the absence of FCS. Supernatants were collected 72 h after transfection, concentrated 8 to 10 times using Centricon 30 spin filters (Millipore, Volketswil, Switzerland), and analyzed by SDS-PAGE and Western blotting.

Northern blotting

Isolation of total RNA, Northern blotting, preparation of ³²P-labeled DNA probes, and membrane hybridization was performed as previously described (19). The membrane was hybridized with the full length hpT49h cDNA and with a 1.3-kbp HindIII fragment of the IL-2R -chain cDNA (pKCR-Tac-2A, provided by Dr. M. Nabholz, ISREC).

RT-PCR analysis

Isolation of total RNA and RT-PCR were done as described (19). Briefly, total RNA extracted from 0.5 x 10⁶-sorted CD3⁺/CD45R0⁺ or CD3⁺/CD45RA⁺ lymphocytes was reverse transcribed into cDNA using Superscript II (Life Technologies). cDNA equivalent to 15 x 10³ cells were used as a template in PCR in the presence of fibroleukin-specific (hupT49F and TS-1R) or ß-actin-specific primers (19). The annealing temperature was 57°C for both primer pairs. hpT49h cDNA was amplified for 40 cycles and ß-actin cDNA for 20 cycles. PCR products were analyzed by 1% agarose gel electrophoresis (21).

Generation of mAbs

The peptide YKSSFKEAKMMIRPKHFKP corresponds to the carboxyl-terminal end of the deduced hpT49h gene product. Fibroleukin sequence was synthesized in an octameric form on a branched amino-terminal core structure (22). Then, 0.5 mg of the peptide and 50 µg of the 6His-FRED recombinant protein (clone 214) were injected s.c. into the Achilles tendon region of BALB/c mice at day 0 (in 50% CFA) and days 3, 6, 9 (in 50% IFA (modified according to 23 . At day 13 the mice were sacrificed, and the enlarged popliteal and inguinal lymph nodes were removed. Eighty million lymphocytes were fused with 20 million Sp2/0 myeloma cells using polyethylene glycol (PEG-1500; Boehringer Mannheim, Mannheim, Germany) according to standard protocols (20). After fusion, the cells were plated into 96-well tissue culture plates (Falcon, Becton Dickinson, Franklin Lakes, NJ) in DMEM or RPMI 1640 supplemented with 10% FCS (Myoclone Super Plus, Life Technologies), 10% Origen Hybridoma Cloning Factor (IGEN, East Jefferson, MD) or 10% BM-Condimed (Boehringer Mannheim), 10 µg/ml hypoxanthine, and 136 µg/ml azaserin (Sigma Chemicals). Growing hybridoma clones were transferred into 24-well tissue culture plates (Falcon) and expanded, and supernatants were collected for screening. The isotype of the anti-fibroleukin mAbs was as follows: 23A6, IgG1; 7B12 and 3F4, IgG3.

ELISA screening

For initial screening, the 19-mer peptide and recombinant 6His-FRED (clone 214) were adsorbed onto Maxisorp ELISA plates (Nunc, Roskilde, Denmark) by overnight incubation at 4°C (0.5 µg/well in PBS). After blocking with 1% BSA, individual hybridoma supernatants were incubated in duplicate wells for 2 h at room temperature. After extensive washing with PBS, mAb binding was revealed using a peroxidase-conjugated goat anti-mouse IgG (1:2000) and tetrametylbenzidine substrate (Sigma Chemicals) according to standard protocols (20). Positive mAbs were further analyzed for their fine specificity toward additional proteins: BSA, plasma fibronectin, plasma fibrinogen, tenascin-C, thioredoxin (Trx), Trx-FRED (clone 129). Ab binding was revealed as above. The chromogenic reaction was measured at 490 nm. Results are expressed in O.D. as a mean of duplicate values ± SD. Blank values were subtracted from the total measured values.

Lymphocyte purification, culture, and activation

PBMC were isolated by discontinuous gradient centrifugation on Histopaque 1.077 (Sigma Chemicals) from venous peripheral blood obtained from healthy donors. T lymphocytes were purified from PBMC by adherence on cell culture plastic and nylon wool columns (Polysciences, Eppelheim, Germany) as previously described (24). Enrichment of large amounts of small resting T cells was obtained with a final centrifugation on 40% Percoll (Pharmacia) (25). The resulting high density T lymphocytes were >95% CD3⁺ and <3% CD16⁺,<3% CD19⁺ as determined by flow cytometry. To isolate large amounts of CD4⁺ and CD8⁺ T lymphocyte subpopulations, magnetically activated cell sorting (MACS, Myltenyi Biotech) was used. T lymphocytes were incubated 30 min on ice with biotinylated mAbs to either CD3, CD4, or CD8. After two washes with cold PBS/0.2% BSA, cells were incubated on ice with streptavidin-conjugated magnetic microbeads. Cells were loaded onto a MACS column and the labeled fraction was positively selected following manufacturer’s instructions. For T lymphocyte activation, PBMC were cultured in complete RPMI 1640 medium with 10% FCS in the presence of 1% PHA (Sigma Chemicals) or plastic immobilized anti-CD3 Ab (TR66, 0.25 µg/ml) and 20 U/ml human rIL-2. Cells were harvested at various times after activation for RNA extraction or metabolic labeling experiments.

Metabolic cell labeling and immunoprecipitation

PBMC (1–4 x 10⁷) or purified T lymphocytes were resuspended (at 1–2 x 10⁷ cells/ml) in Met/Cys-deficient RPMI 1640 medium (ICN, Costa Mesa, CA) supplemented with 2 mM L-Glu, 100 U/ml-100 µg/ml penicillin-streptomycin, and 0.5 mCi of ³⁵S-labeled Met/Cys (Tran-³⁵S-label, ICN). 10% complete RPMI 1640 medium was added for labeling times exceeding 12 h. HepG2 cells were labeled using Met/Cys-free DMEM (ICN). After labeling, culture supernatants were collected and supplemented with 1 mM PMSF (Fluka Chemicals, Buchs, Switzerland), 0.1 µM aprotinin, and 1 µM pepstatin (Boehringer Mannheim). Cells were washed once in PBS and lysed on ice in immunoprecipitation buffer (1% Triton X-100, 0.1% Nonidet P-40, 300 mM NaCl, 100 mM Tris, pH 7.4, 1 mM CaCl₂, and 1 mM MgCl₂) in the presence of protease inhibitors as above. Fresh normal human gut tissue was obtained from a surgically removed colon (Surgery Department of the University Hospital, Lausanne). The mucosal epithelial layer and the lamina propria (1 x 1 cm) were separated from the muscle layer, cut into small pieces, and cultured in 3 ml of DMEM labeling medium containing 0.5 mCi of Tran-³⁵S-label (ICN). After 4 h, the culture supernatant was collected as above.

For protein immunoprecipitation, ³⁵S-labeled culture supernatants or cell lysates were preincubated for 2 h at 4°C with 50 µl of a 1:1 protein A-Sepharose suspension (PAS; Pharmacia, Dübendorf, Switzerland). Then, 250 µl of hybridoma culture supernatants, 5 µg of M2 mAb, or 10 µl of a rabbit anti-human fibrinogen Ig were added to the precleared supernatants and lysates and incubated for 2 h under continuous rotation at 4°C. Ag-Ab complexes were captured by adding 30 µl of a 1:1 PAS suspension and 10 µg of a rabbit anti-mouse Ig during the last h of incubation. The Ag-Ab-PAS complexes were washed 5 times with immunoprecipitation buffer, resuspended in Laemmli sample buffer, and analyzed by SDS-PAGE under reducing or nonreducing conditions (20). Before exposure to film (XAR, Kodak, Rochester, NY), gels were soaked into 1 M sodium salicylate for 30 min and vacuum dried. For neuraminidase treatments, the Ag-Ab-PAS complex was washed three times with 100 mM of sodium acetate, pH 6.5, resuspended in 60 µl of the same buffer in the presence or absence of 0.025 U of neuraminidase (Genzyme, Cambridge, MA), and incubated for 16 h at 37°C.

In some experiments, material analyzed under nonreducing conditions was cut out from the gel and inserted in the slot of a second gel containing reducing sample buffer. After the gel slice was rehydrated, the gel was run and processed as usual.

Western blotting

The material of interest was separated by SDS-PAGE and blotted onto nitrocellulose membrane (Immobilon NC, Millipore, Bedford, MA) following standard protocols (20). Membranes were blocked by overnight incubation at 4°C Tris-buffered saline (TBS, 25 mM Tris, 140 mM NaCl) containing 10% skim dry milk and 0.05% Tween 20. After washing in TBS/0.05% Tween 20, the membranes were incubated with the mAb M2 (1 µg/ml in 10% TBS/10% skim milk), hybridoma supernatants (1:1 diluted in PBS), or control supernatants for 1 h at room temperature. After extensive washing with TBS/0.05% Tween 20, membranes were incubated with a peroxidase-conjugated goat anti-mouse Ig (1:2000 in 10% skim milk, TBS/0.05% Tween 20) for 1 h at room temperature. Bound Abs were revealed using the enhanced chemiluminescence system (ECL, Amersham, Rahn Zürich, Switzerland) and exposed to film.

Flow cytometry analysis and cell sorting

For single staining, cells were labeled in one step with FITC-conjugated anti-CD3, -CD4, -CD8, -CD16, or -CD21 mAbs. For sorting of memory and naive T lymphocytes, PBMC were double labeled with either the anti-CD45R0 or anti-CD45RA mAbs revealed by a PE-conjugated goat anti-mouse Ig and by a FITC-conjugated anti-CD3 mAb. All incubations were done on ice. Labeled cells were analyzed with a FACScan or sorted with a FACStar^Plus (Becton Dickinson).

Immunohistologic procedures

Tissue specimens were obtained from surgically removed tissues and immediately frozen in isopentane cooled in liquid nitrogen. Ten-micrometer sections were cut on a cryostat at -16°C, fixed 5 min in cold acetone, air dried, fixed again in acetone at room temperature, and washed with cold PBS/50 µM thiomersal. Fifty microliters of the 23A6 mAb or Sp2/0 supernatant were incubated for 60 min at room temperature. The sections were washed in PBS/thiomersal, incubated for 25 min with 50 µl of a horse anti-mouse-biotinylated Ab (0.5 µg/ml), washed again in PBS/thiomersal, and incubated with 50 µl of an avidin-peroxidase conjugate solution (100 µg/ml) for 15 min. Sections were stained for 5 min with freshly prepared 3-amino-9-ethyl carbazole (Grade II, Sigma Chemicals, dissolved in 2% dimethylformamide, 0.015% H₂O₂, and 50 mM acetate buffer, pH 5.2). Stained sections were counter-stained in hematoxylin (Polysciences) for 1 min. Sections were viewed and photographed on a Zeiss microscope (Carl Zeiss, Zürich, Switzerland).

For double immunofluorescence, 5-µm frozen sections were immediately fixed in 3% paraformaldehyde in PBS (Sigma Chemicals). The sections were rinsed three times in PBS/1% gelatin (Sigma), quenched for 10 min in 50 mM NH₄Cl, and rinsed three times in PBS/gelatin. Fixed sections were incubated for 30 min successively with FITC-conjugated anti-CD3 mAb (1:5), 23A6 hybridoma supernatant, and PE-conjugated goat anti-mouse Ig (1:100). The sections were washed three times for 5 min in PBS/gelatin between each step, mounted in PBS/glycerol, and viewed and photographed on a Polyvar fluorescent microscope.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of mAbs against the hpT49h gene product

To identify the product of the hpT49h gene, we generated mAbs against synthetic peptides and a recombinant protein fragment. Mice were immunized with a synthetic 19-mer peptide corresponding to the carboxyl-terminal end of the deduced amino acid sequence or the entire FRED domain of the putative protein expressed in Escherichia coli (clone 214, 6His-FRED). Based on the analysis of secondary structure elements of FREDs and on the reported three-dimensional structure of fibrinogen FREDs, the carboxyl-terminal region used to define the synthetic peptide is predicted to be exposed at the surface of the domain (Refs. 26 and 30). Hybridomas were initially screened by ELISA for reactivity against the peptide or the recombinant FRED domain. Positive hybridomas were then tested for lack of reactivity against BSA, fibrinogen, tenascin C, and fibronectin. Several mAbs with these characteristics were identified. Three of these, termed mAbs 23A6, 7B12, and 3F4, were further characterized in Western blotting experiments. All three mAbs recognized the recombinant hpT49h FRED domain in ELISA and Western blotting, but not the proteins fibrinogen, fibronectin, tenascin C, or BSA (Fig. 1 and not shown).

View larger version (53K): [in this window] [in a new window]   FIGURE 1. Reactivity of mAb with fusion proteins containing the fibroleukin FRED domain. a, ELISA. The proteins BSA, human fibronectin (FN), human fibrinogen (FB), Trx, Trx-fibroleukin-FRED fusion protein (cl 129), and fibroleukin-6His-FRED fusion protein (cl 214) were adsorbed to microwells. The reactivity of the mAbs 23A6, 7B12, and 3F4 against these proteins was tested as described in Materials and Methods. Results are expressed in OD units as a mean of duplicate values. b, Western blotting. Human fibronectin (FN), fibrinogen (FB), and total lysate of E. coli cells induced to express Trx or Trx-fibroleukin-FRED (cl 129) were separated by reducing SDS-12% PAGE and transferred onto nitrocellulose membrane. Blots were probed with mAbs 23A6, 7B12, and 3F4. Bound Abs were revealed by peroxidase-conjugated goat anti-mouse Ig and enhanced chemiluminescence detection. The positions and sizes of the molecular mass markers are given.

To demonstrate that these mAbs also recognized the full length protein encoded by the hpT49h gene, we transiently expressed the hpT49h cDNA encoding the putative mature protein in the human embryonic kidney cell line 293T. To allow detection of the protein with an independent anti-FLAG Ab, the cDNA was also expressed with a short sequence encoding the FLAG epitope (DYKDDDDK) (27) fused at the 5' or 3' end. Conditioned cell culture supernatant was collected 72 h after transfection and analyzed in Western blotting experiments. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70-kDa molecular mass (in reducing SDS-PAGE) in the supernatants of cells transfected with the vector containing the hpT49h cDNA, but not in the supernatant of cells transfected with the plasmid alone (Fig. 2, upper panel). The anti-FLAG mAb M2 recognized only proteins containing a FLAG epitope at either the amino- or the carboxyl-terminal end (Fig. 2, upper panel and data not shown). When the same conditioned supernatants were analyzed by nonreducing SDS-PAGE, the immunoreactive material migrated with a molecular mass of approximately 250 to 300 kDa, and the 70-kDa material was no longer detectable (Fig. 2, lower panel).

View larger version (84K): [in this window] [in a new window]   FIGURE 2. Expression of fibroleukin in transiently transfected 293T cells. Conditioned cell culture supernatants of 293T cells transiently transfected with the plasmid pS188 without insert (mock transfection, lane 1), the plasmid pS188 containing the fibroleukin cDNA (lane 2), or the plasmid pS188 containing the fibroleukin cDNA tagged with the FLAG sequence at the amino terminal end (lane 3) were analyzed by reducing (R, upper panel) or nonreducing (NR, lower panel) SDS-PAGE and by Western blotting. The mAbs used in Western blotting are indicated above the blots. The arrowheads indicate the positions of the fibroleukin bands. The positions and sizes of the molecular mass standards are indicated.

Identification of the natural protein encoded by the hpT49h gene

Peripheral blood T lymphocytes express high levels of hpT49hmRNA (19). We therefore used freshly isolated PBMC as a source for the identification of the hpT49h gene product. Freshly isolated PBMC were metabolically labeled overnight, and the culture supernatant was subjected to immunoprecipitation using the mAbs 23A6, 7B12, and 3F4. All three mAbs immunoprecipitated a protein of 70-kDa molecular mass when analyzed in reducing SDS-PAGE (Fig. 3a).

View larger version (49K): [in this window] [in a new window]   FIGURE 3. Immunoprecipitation of fibroleukin produced by PBMC. a, Immunoprecipitation from PBMC supernatant. Culture supernatant from PBMCs (PBMC/S) metabolically labeled for 16 h was subjected to immunoprecipitation with mAbs M2, 23A6, 7B12, and 3F4 or no mAb, as described in Materials and Methods, and analyzed by SDS-PAGE under reducing conditions. Note that bands of 70 kDa are identified by all three Abs. b, Immunoprecipitation from culture supernatant (S) or cell lysate (L) of PBMC pulse-labeled for 2 h. Note that immature forms of fibroleukin (60 kDa) are detected in the cell lysate. c, Neuraminidase treatment. Material immunoprecipitated from the supernatant of 10 x 106 metabolically labeled PBMC was incubated for 16 h at 37°C in the absence (-) or presence (+) of 0.025 U of neuraminidase. d, Lack of cross-reaction between fibroleukin and fibrinogen. The culture supernatants of metabolically labeled PBMCs (PBMC/S, 16 h) or HepG2 cells (HepG2/S, 4 h) were subjected to immunoprecipitation with mAb 23A6 or an anti-human fibrinogen (a-FB) antiserum. The positions of the mature fibrinogen A-, Bß-, and -chains are indicated. In all experiments, immunoprecipitated material was analyzed by reducing SDS-PAGE and revealed by autoradiography. The black arrowhead indicates mature fibroleukin, the white arrowhead indicates desialylated fibroleukin, and arrows indicate immunoreactive material in the lysate. Asterisks indicate nonspecifically coprecipitated material.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Fibroleukin production and mRNA expression by peripheral blood T lymphocyte subsets. a, The culture supernatants of MACS-sorted and metabolically labeled CD3+, CD4+, and CD8+ peripheral blood T lymphocytes (20 x 106 cells) were subjected to immunoprecipitation as indicated. Immunoprecipitated material was analyzed by SDS-7.5% PAGE under reducing conditions followed by autoradiography. The arrowhead indicates fibroleukin, and the asterisks indicate nonspecific coprecipitated material. b, Detection of fibroleukin mRNA. cDNA derived from FACS sorted CD3+/CD45R0+ (memory) and CD3+/CD45RA+ (naive) T lymphocytes was amplified with primers specific for fibroleukin (40 cycles) or ß-actin (20 cycles), respectively. PCR products were analyzed by 1% agarose gel electrophoresis. SLA cells were used as a positive control. Arrowheads indicate the fibroleukin and ß-actin amplification products (529 and 700 bp, respectively). M, DNA size markers (type IV, Boehringer Mannheim).

The hpT49h gene product is secreted as a disulfide-linked complex

To test whether the natural hpT49h gene product is also secreted as a disulfide-bonded complex, we analyzed material immunoprecipitated from the supernatant of PBMC by nonreducing SDS-PAGE. As shown in Figure 4a, under nonreducing conditions this material migrated with 250 to 300 kDa molecular mass and is visible at the top of the gel. To demonstrate that this material consisted of the 70-kDa protein, the corresponding region of the gel was cut out and loaded onto a second gel for analysis under reducing conditions. As shown in Figure 4b (lane 2), the characteristic 70-kDa protein is visible after reduction. The relatively large amount of the 65-kDa protein may be due to degradation of the protein during reelectrophoresis. Material reanalyzed under nonreducing conditions is visible, again, at the top of the gel (Fig. 4b, lane 1). These data demonstrate that the natural hpT49h gene product is secreted as a disulfide-bonded complex.

View larger version (58K): [in this window] [in a new window]   FIGURE 4. Fibroleukin is secreted as a disulfide bonded protein complex. a, Material immunoprecipitated with the mAb 23A6 from the supernatant of 20 x 106 metabolically labeled PBMC was analyzed in nonreducing SDS-5% PAGE. b, The material indicated by the arrow in panel a was excised and reanalyzed in a second gel under nonreducing (NR, lane 1) or reducing (R, lane 2) conditions. Material immunoprecipitated by mAb 23A6 and analyzed directly under reducing conditions is shown as a comparison (lane 3). The arrowheads indicate fibroleukin. Positions and sizes of the molecular mass standards are indicated next to each autoradiogram.

Peripheral blood resting CD3⁺, CD4⁺, and CD8⁺ T lymphocytes secrete fibroleukin

We previously reported the expression of fibroleukin mRNA in peripheral blood CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T lymphocytes (19). To demonstrate protein production and secretion by T lymphocytes, we purified large amounts of CD3⁺, CD4⁺, and CD8⁺ T lymphocyte subsets by magnetically activated cell sorting (MACS) for use in metabolic labeling and immunoprecipitation experiments. The purities of the MACS-purified T lymphocytes were 98, 98, and 85% for the CD3⁺, CD4⁺, and CD8⁺, subsets respectively, as revealed by subsequent FACS analysis (data not shown). Cells were obtained from the same donor, purified in parallel procedures, and equal numbers were used in each experiment. As shown in Figure 5a, mAb 23A6 immunoprecipitates fibroleukin from the supernatant of all three T lymphocyte subsets, implying that both CD4⁺ and CD8⁺ T lymphocyte subsets secrete the protein.

We also tested whether fibroleukin is present on the surface of circulating T lymphocytes. Freshly isolated PBMC were double stained for the T lymphocyte markers CD3, CD4, or CD8 and with the mAb 23A6. Fibroleukin staining was not detected on CD3⁺, CD4⁺, or CD8⁺ T lymphocytes (data not shown).

Fibroleukin mRNA is preferentially expressed in memory T lymphocytes

Memory and naive T lymphocytes differ in the expression of several genes and their products, including adhesion receptors and cytokines (28). To examine whether the fibroleukin gene may also be differentially expressed in these two T lymphocyte subsets, we analyzed fibroleukin mRNA-expression by RT-PCR in FACS-sorted lymphocytes. A fibroleukin amplification signal was consistently obtained on cDNA derived from CD3⁺/CD45R0⁺ (memory) T lymphocytes (Fig. 5b). In contrast, very weak or no fibroleukin signal was observed when cDNA derived from CD3⁺/CD45RA⁺ (naive) T lymphocytes was amplified.

Fibroleukin production is rapidly lost in culture and is not induced by T cell activation

Culture and activation of T lymphocytes can alter the production of many T lymphocyte proteins. To test the effect of activation and proliferation on fibroleukin secretion, PBMC were cultured in the absence or in the presence of the polyclonal T lymphocyte mitogen PHA. After 3 days of culture, unstimulated PBMC lost most of their capacity to produce fibroleukin compared with freshly isolated PBMC (Fig. 6a). PBMC stimulation with PHA had little or no effect on the loss of fibroleukin production. In contrast, in the presence of IFN-, fibroleukin production remained at higher levels. We also activated T lymphocytes with plastic-immobilized anti-CD3 mAb, which more closely resembles physiologic T cell activation. Cells were cultured in the presence of IL-2 for up to 8 days, and aliquots were collected for metabolic labeling and RNA extraction at days 2, 4, 6, and 8 after activation. The result, shown in Figure 6b, confirmed that T lymphocyte activation had no effect on the loss of fibroleukin secretion by T lymphocytes. To investigate whether the decrease in fibroleukin secretion was transcriptionally regulated, we analyzed fibroleukin mRNA levels by Northern blot hybridization. The result, shown in Figure 6c, demonstrates that fibroleukin mRNA level decreases with time, paralleling the loss of protein production and secretion. The same blot was successively hybridized with the IL-2R -chain cDNA to demonstrate T lymphocyte activation.

View larger version (52K): [in this window] [in a new window]   FIGURE 6. Fibroleukin production and mRNA expression in cultured PBMC. a, Fibroleukin expression in cells treated with PHA or IFN-. Fibroleukin was immunoprecipitated from the supernatant of PBMC metabolically labeled immediately after purification (top panel), or after 3 days of culture in the absence of stimuli (second panel from top), or in the presence of PHA (third panel) or IFN- (1000 U/ml, fourth panel). The mAbs used for immunoprecipitation are indicated above each lane. b, Peripheral blood T lymphocytes were activated either by PHA or by plastic-immobilized anti-CD3 Ab in the presence of IL-2 (20 U/ml). Cells (107) were metabolically labeled for 16 h before (day 0) or after activation (day 2, 4, 6, and 8). Supernatants were subjected to immunoprecipitation with mAb 23A6 as indicated and analyzed by reducing SDS-7.5% PAGE followed by autoradiography. c, Northern blot analysis of fibroleukin mRNA expression. 15 µg of total RNA obtained before (0) and at day 2, 4, 6, and 8 after PBMC stimulation with PHA was probed for fibroleukin and IL-2R -chain mRNA by Northern blot hybridization. The ethidium bromide staining of the filter before hybridization is shown to demonstrate equal RNA loading.

Fibroleukin production in colon mucosa

Since the hpT49h/fibroleukin cDNA was cloned from a human intestine cDNA library (19), we examined gut tissue to demonstrate fibroleukin production in vivo. The mucosa of a surgically removed colon was cultured and metabolically labeled for 4 h. The culture supernatant was then subjected to immunoprecipitation with mAb 23A6. The characteristic 70-kDa protein band was readily detected, thus demonstrating fibroleukin production in intestinal mucosa (Fig. 7a).

View larger version (75K): [in this window] [in a new window]   FIGURE 7. Fibroleukin expression in colon mucosa. a, Colon mucosa (1 x 1 cm) was cut into small pieces and metabolically labeled in culture for 4 h with 0.5 mCi of Tran-35S-label. Culture supernatant was collected for immunoprecipitation with the 23A6 mAb. Immunoprecipitated material was analyzed by SDS-7.5% PAGE and autoradiography. Arrowhead indicates fibroleukin. Position and size of the molecular mass standards are indicated. b–i, Immunolocalization of fibroleukin in colon lamina propria. Frozen sections of colon tissue were processed for immunohistochemistry with mAb 23A6 (c, d, e) or control mAb supernatant (b) and for double immunofluorescence with FITC-conjugated anti-CD3 mAb (f, h) and mAb 23A6 revealed by goat anti-mouse-PE (g, i). Fibroleukin staining is mostly localized in the ECM, surrounding cells present in the upper portion of the lamina propria mucosae, while deeper regions and isolated lymph follicles (c, arrowheads) were negative. The double immunofluorescence staining shows localization of fibroleukin in areas rich in CD3+ T lymphocytes (f and g, dots) and also in close proximity to single CD3+ cells (h and i, black arrowheads). Some CD3+ T lymphocytes, including intraepithelial T lymphocytes, and their surrounding ECM are negative (h and i, white arrowhead), The goat anti-mouse-PE control was negative (not shown). Bars: 200 µm, b–d, f, and g; 50 µm, e, h, and i.

To determine the spatial association between T lymphocytes and fibroleukin deposition, colon tissue sections were double stained with the mAb 23A6 and with an anti-CD3 mAb. These experiments revealed that fibroleukin is present in CD3⁺T lymphocyte-rich areas (Fig. 7, f–i). While most of the fibroleukin staining was observed in the ECM in close proximity to CD3⁺ lymphocytes, there were, however, areas infiltrated by CD3⁺ cells that were negative for fibroleukin. We observed no fibroleukin staining associated with intraepithelial lymphocytes.

We conclude that a subset of CD3⁺ T lymphocytes localized in the upper portion of the lamina propria produces fibroleukin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this paper, we report the identification and the initial characterization of human fibroleukin, the fibrinogen-like protein encoded by the hpT49h transcript.

To identify fibroleukin, we used mAbs generated by immunizing mice with a synthetic peptide based on the deduced amino acid sequence and a recombinant protein corresponding to the FRED of the hpT49h gene product expressed in E. coli. Three mAbs, 23A6, 7B12, and 3F4, recognize a 70-kDa protein secreted as a disulfide-bonded complex of 250 to 300 kDa by freshly isolated PBMC and highly purified peripheral blood T lymphocytes. We consider this protein to be the product of the hpT49h gene for the following reasons.

First, the Abs used for its detection are specific for the hpT49h gene product. They recognize the recombinant FRED expressed in E. coli as well as the full length recombinant protein expressed in mammalian cells from the hpT49h cDNA, but not the proteins fibrinogen and tenascin C, which contain highly homologous FREDs. Second, the main biochemical characteristics of fibroleukin are consistent with those predicted by the analysis of the deduced amino acid sequence: it is a secreted protein (as proposed by the presence of a putative signal peptide), the observed size of 70 kDa is in good agreement with the calculated molecular mass (65 kDa) (19), and it forms a disulfide-bonded complex, as expected from its homology, to the fibrinogen ß- and -chains. Third, recombinant fibroleukin expressed from the hpT49h cDNA in mammalian cells have the same molecular mass and ability to form a disulfide-bonded complex as natural fibroleukin.

Throughout these experiments, we found no evidence for the presence of additional proteins in the complex, suggesting that the complex consists only self-associated fibroleukin. The observed molecular mass of 250 to 300 kDa is indicative of a complex containing four fibroleukin subunits. A tetrameric complex consisting of two dimers is in good agreement with the predicted structural features of fibroleukin. Fibroleukin dimers can occur through coiled-coil interactions within the amino-terminal half of the molecule, which are then stabilized by interchain disulfide bonds similar to those present in the fibrinogen complex. Association of two dimers to form the tetrameric complex can be mediated by disulfide bonds involving the most amino-terminal Cys residues. The tetrameric complex would consist of a central, elongated structure flanked by two more globular FREDs at each end, similar to the structure of the fibrinogen complex (29, 30). Alternatively, it is also possible that the four fibroleukin subunits associate to form a tetrameric coiled-coil structure, thus generating a complex having all four FREDs at one end and the elongated coiled-coil region at the other end. The determination of the precise structure of the complex will require further biochemical and biophysical analysis.

Our results demonstrate fibroleukin secretion by nonstimulated PBMC and by highly purified CD4⁺ and CD8⁺ peripheral blood T lymphocytes. We have detected mRNA expression and protein production by several EBV-B-transformed B cell lines (Ref. 19 and our unpublished results). Expression of pT49 mRNA by murine macrophages was demonstrated in two recent reports (31, 32). From these data, it appears that fibroleukin expression is not limited to cytolytic T lymphocytes as initially proposed (18). The production of fibroleukin by circulating peripheral blood T lymphocytes is in apparent contrast with the restricted deposition of fibroleukin observed in vivo. This apparent discrepancy, however, can be explained by the fact that T lymphocytes are in continuous recirculation between different body compartments and the peripheral blood (33). The fibroleukin secreted by peripheral blood T lymphocytes may be produced by cells circulating through specific tissue sites, such as the gut lamina propria. Since lamina propria lymphocytes consist mostly of memory T lymphocytes (34), this hypothesis is in good agreement with the preferential expression of fibroleukin mRNA in peripheral blood memory T lymphocytes as opposed to naive T lymphocytes.

Expression of the fibroleukin gene in T lymphocytes appears tightly regulated and is likely to reflect differences in the functional and/or differentiation states of the cell. Activated and proliferating T lymphocytes express reduced amounts of fibroleukin compared with freshly isolated T lymphocytes. The presence of the fibroleukin in the upper region of the lamina propria, but not in the mucosa-associated lymph follicles, correlates with the anatomically and functionally different compartments of the gut-associated lymphoid tissue (GALT). Lymphoid cells present in the organized GALT, Peyer’s patches, isolated lymph follicles, and mesenteric lymph nodes constitute the afferent component of the GALT in which Ag presentation, T and B lymphocyte activation, and proliferation occur. In contrast, the lymphocytes that are diffusely distributed within the lamina propria represent the efferent component of the GALT in which effector functions, such as IgA production by plasma cells and T lymphocyte helper and cytolytic functions, occur (35, 36). Moreover, T lymphocytes present in the organized GALT consist of both memory and naive subpopulations, while the T lymphocytes present in the lamina propria consist almost exclusively of memory cells (33, 34).

Our data indicate that T lymphocyte activation per se does not induce fibroleukin secretion, suggesting that other mechanisms may be implicated in its regulation. Locally produced cytokines are prime candidate molecules; many cytokines, such as IL-4, IL-5, IL-6, TGF-ß, or IFN- (35, 37), are normally produced in the intestinal mucosa. In line with this hypothesis, we found that IFN- induces production of fibroleukin in PBMC, and Lafuse and colleagues have previously reported that IFN- induces pT49 mRNA expression in murine peritoneal exudate cells (32).

What is the function of T lymphocyte-derived fibroleukin? Its structural similarity with fibrinogen and its expression features reported here allow us to hypothesize about some possible functions. Fibroleukin may posses adhesion-related activities. A large number of lymphocytes are thought to recirculate through the intestinal lamina propria. Fibroleukin may participate in mediating extravasation or migration of memory T lymphocytes through the gut by acting as an adhesion-promoting substrate. The extravasating and infiltrating T lymphocytes may secrete fibroleukin, which could be used as a specific adhesion substrate by T lymphocytes themselves or by other cells. To support adhesion or migration, fibroleukin may require association, at least transiently, with insoluble ECM components. Alternatively, fibroleukin may remain soluble and could serve as a bridging molecule to promote cell-cell contacts. It may facilitate T lymphocyte-endothelial cell interaction during extravasation, or T-B lymphocyte interaction during the helper function. It may also mediate attachment to target cells for cytolytic activity. An extension of the analogy between fibrinogen and fibroleukin suggests that integrin-type adhesion receptors may be involved in mediating fibroleukin-cell interactions. The absence of an RGD motif does not rule out this possibility, since many integrin-ligand interactions occur in an RGD-independent manner (38).

In addition to an adhesive function, fibroleukin may also possess cytokine-like activity. The lamina propria is an immunologically active place, and the only tissue in which large amounts of plasma cells, mostly secreting IgA, are present under physiologic conditions. T and B lymphocytes in the lamina propria must therefore be continuously replaced. This implies that plasma cell precursors and fresh T lymphocytes with helper activity have to accumulate and differentiate continuously. Cytokines and chemokines, in addition to direct cellular contacts, play a key role in controlling these events (35). The diffuse and gradient-like staining pattern of fibroleukin in the lamina propria and its close association with infiltrating T lymphocytes are characteristics compatible with a cytokine-like function. Fibrinogen itself has been reported to exert a mitogenic activity on B lymphocytes by delivering signals via specific cell surface receptors (39, 40).

It was recently shown that the murine pT49 gene is expressed in macrophages stimulated by IFN- (32) or mouse hepatitis virus strain 3 (MHV-3) infection (31). Parr and colleagues (31) have reported the molecular cloning of the murine pT49 transcript by screening an expression cDNA library in E. coli using a mAb, termed 3D4.3, that was previously shown to block a procoagulant activity produced by MHV-3-infected macrophages (41, 42). The authors suggested that the pT49 gene encodes a macrophage-derived prothrombinase. There was, however, no direct evidence that the pT49/musfibpl gene product itself has prothrombinase activity. It appears unlikely that fibroleukin secreted by lamina propria T lymphocytes acts as a prothrombinase, because our results suggest that nonactivated memory T lymphocytes normally secrete fibroleukin under conditions that are not likely to stimulate clot formation.

In conclusion, we have identified fibroleukin as the product of the hpT49h gene and have initiated its characterization. In a phylogenetic perspective, fibroleukin may be considered a specialized form of fibrinogen. Fibroleukin appears to have separated from a fibrinogen-precursor gene before the appearance of the three fibrinogen chains (19). While fibrinogen has acquired additional structural complexity and has evolved into a multifunctional molecule that plays a central role in hemostasis and tissue repair throughout the body, fibroleukin appears to have retained a simpler structure and to have evolved into a molecule with activities possibly related to immune functions at tissue sites. Current experiments are aimed at identifying and characterizing these functions, as well as analyzing fibroleukin expression and distribution in human tissues under normal and pathologic conditions.

	Acknowledgments

 
We thank Drs. J. Tschopp, P. Schneider, M. Nabholz, and S. Abrignani for providing reagents; Ms. F. Mourali, D. Minaidis, and M. G. Bieler for technical assistance; Mr. P. Zaech for FACS analysis and cell sorting; and M. Allegrini and P. Dubied for artwork. We also thank Drs. Jean-Charles Cerottini and Ferdy Lejeune for helpful discussions and support.

	Footnotes

 
¹ This work was supported in part by Grant 31-40286.94 from the Swiss National Science Foundation (to C.R.); Fellowship 92-37 from the American Heart Association, California Affiliate (to C.R.); Fonds de Recherches sur les Lymphomes Malins Charles Veillon, Lausanne; and Grant CA53250 from the National Institutes of Health (to R.P.). C.R. is the recipient of a SCORE A Career Development Award from the Swiss National Science Foundation.

² Address correspondence and reprint requests to Dr. Curzio Rüegg, Laboratoire du Centre Pluridisciplinaire d’Oncologie (CPO), c/o ISREC, 155 Chemin des Boveresses, CH-1066 Epalinges, Switzerland. E-mail address:

³ Abbreviations used in this paper: ECM, extracellular matrix; PAS, protein A-Sepharose; MACS, magnetically activated cell sorting; FRED, fibrinogen-related domain; GALT, gut-associated lymphoid tissue; Trx, thioredoxin; PE, phycoerythrin.

Received for publication October 15, 1996. Accepted for publication March 2, 1998.

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