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Human polymorphonuclear neutrophils express a B7-1-like molecule

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  • 1. Human polymorphonuclear neutrophils express a B7-1-like molecule Anja Windhagen, Susanna Maniak, Andreas Gebert,* Isabel Ferger, Ulrich Wurster, and Fedor Heidenreich Laboratory of Neuroimmunology, Department of Neurology, and *Center of Anatomy, Medical School Hannover, Germany Abstract: Polymorphonuclear neutrophils (PMN) of B7-2. The question of whether the two costimulatory are part of the innate immune system and are molecules B7-1 and B7-2 have different or overlapping func- first-line effector cells in acute inflammatory re- tions is unresolved as yet [13–16]. sponses. On activation PMNs secrete cytokines and The interactions between PMNs and T cells might be oxygen metabolites that might be involved in the important in human inflammatory diseases, e.g., inflammatory regulation of the acquired immune response. We central nervous system (CNS) diseases like bacterial or viral show here that peripheral blood PMNs constitu- meningitis, in which PMNs are the predominant cell type in tively express a B7-1-like molecule as detected by early phases of the immune response. However, PMNs can also immunostaining with several B7-1 antibodies. Re- be found in inflammatory lesions of autoimmune diseases like verse transcriptase-polymerase chain reaction us- rheumatoid arthritis or experimental autoimmune encephalomy- ing three sets of primers spanning different regions elitis (EAE), an animal model for multiple sclerosis (MS) of B7-1 indicate dissimilarities at the mRNA level. [17, 18]. B7-1 mRNA is expressed in bone marrow cells and We examined the expression of costimulatory molecules lipopolysaccharide (LPS)-stimulated but not in un- B7-1 and B7-2 in inflammatory CSF specimens from patients stimulated PMNs. The B7-1-like molecule is local- with bacterial meningitis. It was surprising to find that in all ized to the cytoplasmic granules and translocated to CSF samples staining of PMNs with the monoclonal Ab (mAb) the cell surface after stimulation with LPS or 104 (B7-1, mAb 104 antigen) was detected. We then investi- interleukin-12 in some donors. Binding of CTLA4-Ig gated expression and regulation of B7 molecules in PMNs. suggests that the B7-1-like molecule can interact with functional B7 ligand and might be important in the immunobiology of PMNs. J. Leukoc. Biol. 66: MATERIALS AND METHODS 945–952; 1999. Patients Key Words: intracellular · inflammatory responses · bone marrow Cerebrospinal fluid (CSF) was obtained from eight patients who underwent cells routine diagnostic lumbar puncture for bacterial meningitis at the Department of Neurology, Medical School Hannover, during November 1996 and August 1997 (Table 1). Patients gave informed consent that samples be used for research purposes after clinical diagnostic tests were completed. None of the INTRODUCTION patients had received antibiotic treatment before the experiments. Patients 3 and 4 had tuberculous meningitis, patient 7 had Streptococcal meningitis. In Polymorphonuclear neutrophils (PMNs) are first-line effector the other patients CSF culture for bacteria was not revealing. cells in microbial infections and on stimulation produce a variety of bioactive substances, e.g., enzymes, cytokines, and Reagents reactive oxygen metabolites, that mediate cytotoxicity and Primary antibodies used were anti-B7-1 (clone mAB104, IgG1, Immunotech, inflammatory reactions in the tissue [1–3]. Recently several Marseille, France; clones EW3.1F1.F5, IgG2b, and EW3.4B2.C4, IgG2a, gifts reports suggested that PMNs may be capable of presenting from Genetics Institute, Cambridge, MA; clone L307.4, IgG1, from Becton Dickinson, and clone P1.H5.A1.A1, IgG1, from Ancell, Bayport, MN), antigen to T lymphocytes because they can be induced to anti-B7-2 (clone FUN-1, IgG1, PharMingen, San Diego, CA), anti-CD16 (clone express major histocompatibility complex II (MHC II) mol- 3G8, IgG1, PharMingen), and nonspecific mouse IgG1, IgGb, and IgG2a ecules [4]. Eosinophils from interleukin-5 (IL-5) transgenic isotype controls (DAKO, Glostrup, Denmark). CTLA4-Ig was obtained from mice express both B7-1 and B7-2 [5] and expression of B7-2 Ancell. Human B7-1/IgG-Fc fusion protein (a gift from Genetics Institute) was was also found on eosinophils in inflammatory skin lesions in used as positive control for Western blot. mice [6]. The B7- molecules deliver the most potent costimula- tory signals to T cells [7] and simultaneous blockade of these molecules causes complete inhibition of T cell responses [8, 9]. Correspondence: Anja Windhagen, Department of Neurology, Medical In humans B7-2 but not B7-1 is expressed on resting B cells, School Hannover, 30623 Hannover, Germany. E-mail: Windhagen.Anja@MH- dendritic cells, monocytes, and microglia [7, 10–12]. After Hannover.de stimulation, these cells also express B7-1 and increase the level Received March 1, 1999; revised August 2, 1999; accepted August 3, 1999. Journal of Leukocyte Biology Volume 66, December 1999 945
  • 2. TABLE 1. Patients with Bacterial Meningitis (Bio-Rad, Munich, Germany) and fixed in 4% PFA (Sigma) for 30 min. After blocking with 2% fetal calf serum (FCS) washing was done in EBSS. Cells were Disease mAb 104 incubated with primary antibody in EBSS/10% human AB serum (Sigma) for CSF PMNs, Monocytes, Lymphocytes, duration, intra- mAb 104 30 min at room temperature followed by secondary antibody [fluorescein Patient cells/µL % % % days cellular surface, % isothiocyanate (FITC)-or phycoerythrin (PE)-conjugated goat-anti-mouse IgG1 1 120 91 2 7 1 — or IgG, Southern Biotechnology, Birmingham, AL] for another 30 min. For cytoplasmic staining 0.1% saponin (Sigma) was added to the washing and 2 2120 94 1 5 1 — staining buffers during the whole process. Slides were covered with AquaPoly- 3 171 54 4 42 24 2, 5 mount (Polysciences, Warrington, PA) and viewed under a confocal microscope. 4 213 47 9 44 10 — 5 330 63 11 26 9 — 6 48 51 9 40 14 — Flow cytometry 7 1336 12 22 66 19 79 8 1213 98 0 2 1 — For flow cytometry the same primary and secondary antibodies as above were used after fixation of cells with 4% PFA for 15 min. Cells were stained with or Shown are the total CSF cell count, the percentages of different cell types, without 0.1% saponin buffer that was supplemented with 10% human AB and the percentages of PMNs that had surface staining with mAb 104 (B7-1). In serum to block unspecific antibody binding for 15 min for each primary and all samples intracellular staining of PMNs with mAb 104 was detected. secondary antibody at room temperature and analyzed on a FACScan (Becton Dickinson). Mean fluorescence intensities were determined from data histo- grams with the use of CellQuest software. As positive control for B7 staining, Cytokines used for stimulation were IL-8 at 5 µg/mL, interferon- (IFN- ) at the Epstein-Barre virus (EBV)-transformed B cell line LE509 (a gift from R. 1000 U/mL, and IL-12 at 50 ng/mL all from R & D Systems (Minneapolis, MN). Schmidt, Hannover), which expresses both B7-1 and B7-2 on the cell surface, Other cytokines used were recombinant IL-1 and IL-6 from Tebu (Frankfurt, was used. Germany) at 100 ng/mL, granulocyte colony-stimulating factor (G-CSF) from Amgen (Thousand Oaks, CA) at 100 ng/mL, GM-CSF from PeproTech, Inc. (Rocky Hill, NY) at 30 ng/mL, and tumor necrosis factor (TNF- ) from Alexis Reverse transcriptase-polymerase chain Corp. (Grunberg, Germany) at 1000 U/mL. reaction (RT-PCR) Other reagents used were PMA at 10 ng/mL, N-formyl-methionyl-leucyl- phenylalanine (fMLP) at 10-7 M, and lipopolysaccharide (LPS) at 10 µg/mL, all Total cellular RNA was extracted from 2 106 cells and cDNA was prepared as from Sigma (Deisenhofen, Germany). described previously [20, 21]. Each 25-µL PCR contained 5 µL cDNA, 0.5 µg of forward and 0.5 µg reverse primers, 1.2 U of Taq polymerase, and 10 µL of a mix Cell purification and culture of dNTPs and Taq buffer that was prepared as a master mix for each set of samples. Thermal cycling was done at 94°C for 1 min, 55 or 60°C for 1 min, and PMNs were isolated from the peripheral blood of healthy donors as previously 72°C for 1.5 min for 28–35 cycles. Primer sequences for -actin and IL-12 described [19]. In brief, blood was layered over Ficoll and peripheral blood were as described previously [20]: forward primer B7-1, 5 AAC TCg CAT CTA mononuclear cells (PBMCs) were removed. PMNs were separated from red CTg gCA AAA ggA gAA 3 ; reverse primer B7-1, 5 ggg AAA CTg TTg TgT blood cells (RBCs) by sedimentation in 3% hydroxyethyl starch HES 450/0.7 TgA Tgg CAT TTA 3 ; forward primer B7-2, 5 gTA TTT Tgg CAg gAC CAg gA (Fresenius, Bad Homburg, Germany). Remaining RBCs were lysed with H2O. 3 ; reverse primer B7-2, 5 gCC gCT TCT TCT TCT TCC AT 3 . The resulting cell population was stained with the naphthol-AS-D-chloroacetate To determine B7-1 mRNA expression, two additional primer pairs (B7-1a (NCAE) kit from Sigma to determine monocyte contamination and percentage of and B7-1b) were used that amplify regions from the amino- or carboxy-terminal eosinophils in the preparation. Here neutrophils are stained red, whereas end of the B7-1 cDNA (accession number M27533). B7-1a forward 5 ATg ggC eosinophils are not stained. CAC ACA Cgg Agg-3 , B7-1a reverse 5 Tgg gCg CAg AgC CAg gAT CA-3 , PMNs were suspended in RPMI-1640 (Sigma) supplemented with 10% FCS B7-1b forward 5 ACT ggC AAA Agg AgA AAg AAA-3 , B7-1b reverse 5 ATA (GIBCO, Eggenstein, Germany) and 1% HEPES (Sigma) and cultured at 37°C CAg ggC gTA CAC TTT CC-3 . for up to 44 h in 5-mL polypropylene tubes (Greiner, Nurtingen, Germany) at PCR products were separated on 1.5% agarose gels, stained with ethidium 107/mL in the presence of cytokines or LPS. Stimulation with PMA or fMLP was bromide, and analyzed on a video imaging system (Eagle-Eye, Stratagene). performed for 25 min. To determine cell viability the Trypan blue exclusion test was used. To inhibit shedding of B7 from the cell surface by protease activity a protease inhibitor cocktail (Roche Molecular Biochemicals) 1 tablet/10 mL was Immunoblotting added to the culture medium. Expression of B7-1 and B7-2 was examined after Whole-cell lysates were prepared from 5 106 untreated and PMA- or 15, 30, 60, and 120 min and after 8, 16, 20, and 44 h on the surface and in the fMLP-stimulated PMNs by homogenizing in 0.3 mL PBS/0.1% Tween contain- cytoplasm by FACS analysis and immunohistochemistry. For RT-PCR, RNA ing a protease inhibitor cocktail (Roche Molecular Biochemicals) 1 tablet/10 was extracted after 6, 12, and 20 h of stimulation. mL. Sixty micrograms of protein from cell lysates or four micrograms protein Bone marrow was obtained from two healthy subjects who donated bone from cell supernatant was loaded in each lane on 7.5–18.5% T gradient gels marrow for transplantation purposes at the Department of Hematology, Medical and run overnight under non-reducing conditions. Recombinant B7-1/IgG-Fc School Hannover, after informed consent. Bone marrow cells were isolated by fusion protein (0.5 ng) was used as positive control. The separated proteins were Ficoll separation to remove RBCs and granulocytes. transferred electrophoretically to 0.2-µm nitrocellulose membranes in a tank blot apparatus. The membranes were blocked with 5% skimmed milk and Immunocytochemistry incubated overnight with 0.4 µg/mL mAb 104 (Immunotech). After three For APAAP staining, cytospins were fixed in acetone for 2 min. Primary washes with TBS-Tween a biotinylated rabbit anti-mouse antibody (Dako, antibody was added in phosphate-buffered saline (PBS)/1% bovine serum E0354 at 1:2000) was added and developed for 45 min using streptavidin/ albumin (BSA) for 30 min, and fixed for 2 min in 0.04% glutaraldehyde (Merck, alkaline phosphatase complex and BCIP/NBT as color substrates. Darmstadt, Germany). Rabbit anti-mouse IgG was added for 30 min followed by incubation with APAAP mouse monoclonal antibody, both from DAKO. Bridging antibody and APAAP complex were each repeated for 15 min. Substrate solution (Fast-red tablets, KEM EN TEC, Copenhagen, Denmark) RESULTS was added in 0.1 M Tris buffer for 15 min. Counterstaining was done with Hemalum solution (Merck) for 2 min. Slides were mounted with Aquamount Neutrophils in the CSF from bacterial meningitis Improved (BDH Laboratory Supplies, Poole, UK) and examined by light microscopy. The fixation with acetone allows for intracytoplasmic staining. To investigate expression of costimulatory molecules on CSF For immunofluorescence cells were transferred to Bio-Rad adhesion slides cells from patients with bacterial meningitis, samples were 946 Journal of Leukocyte Biology Volume 66, December 1999 http://www.jleukbio.org
  • 3. Expression of B7 molecules in peripheral blood PMNs To determine whether expression of B7-1 is a common feature of PMNs or is present only in PMNs in inflammatory reactions we next examined PMNs from the peripheral blood of healthy donors. We performed surface and intracytoplasmic staining for B7-1 and B7-2 mAbs. The PMN preparation obtained consisted of 96–99% neutrophils as determined by CD16 expression and NCAE staining, the contaminating cells being eosinophils. Monocytes and lymphocytes were not detectable. PMNs from the peripheral blood expressed the mAb 104 antigen (B7-1) in the cytoplasm, whereas B7-2 expression was not detectable (Fig. 3). Staining was also obtained by binding of two other antibodies to B7-1 (EW3.1 and P1.H5.A1.A1) as well as CTLA4-Ig, the functional ligand for B7-1 and B7-2. However, Fig. 1. Immunocytochemistry of CSF cells from a patient with bacterial the B7-1 antibodies L307.4 and EW3.4 did not stain PMNs meningitis (acute phase, PMN percentage 98%). (A) mAb 104, (B) B7-2 mAb. (Fig. 3). This staining pattern was defined as B7-1-like. All PMNs show positive staining with mAb 104, but not B7-2 mAb. B7-2 Eosinophils also showed positive staining with mAb 104 (not staining, however, is detectable on CSF monocytes. The same results were shown), however, further study of eosinophils with immunofluo- obtained also from the other CSF specimen tested. stained with the B7-1 (mAb 104) and B7-2 mAbs. In all samples both intracellular and surface staining of CSF cells was performed. PMNs from all CSF samples showed intracellular staining with mAb104 (B7-1; Fig. 1A) but not B7-2. Lympho- cytes and monocytes present in some CSF samples (Table 1) did not express B7-1. In addition, PMNs from CSF of two patients (one with tuberculous meningitis and one with streptococcal meningitis) that both were in the subacute phase of the disease showed surface expression of B7-1 as detected by staining with mAb 104 (Fig. 2). B7-2 was detected on CSF monocytes only (Fig. 1B). Fig. 3. Expression of B7-1-like molecule in PMNs. (A–F) Cytoplasmic staining of PMNs with different B7-1 Abs (mAb 104, EW3.1, P1.H5.A1.A1, L307.4) as well as B7-2 and CTLA4-Ig. Positive staining of PMNs is seen with Fig. 2. Surface staining with mAb 104 of CSF neutrophils from two patients Abs 104, P1.H5.A1.A1, EW3.1, and CTLA4-Ig but not with B7-2 mAb or with bacterial meningitis. Neutrophils were gated by forward/sideward scatter L307.4. B7-1 Abs EW3.4 also did not stain PMNs (not shown). (G, H) Surface characteristics. (A) patient 7, (B) patient 3 (see Table 1). In (A) there were 79% staining of EBV cells with mAb 104 and EW3.4. Compared with mAb104 and of CSF neutrophils expressing B7-1 on the cell surface; in (B) the percentage of the other B7-1 antibodies used, EW3.4 only showed weak staining of EBV positively stained neutrophils was 2.5%. cells. Windhagen et al. B7-1 expression in human neutrophils 947
  • 4. rescence was not successful due to high nonspecific staining To further study the regulation of the B7-1 like molecule in and autofluorescence. PMNs we examined whether surface expression of the B7-1- EBV cells were used as positive control because these cells like molecule could be induced on PMNs from the peripheral constitutively express high levels of B7-1 and B7-2. All B7-1 blood of healthy donors. Because most intense staining was antibodies used in this study showed positive staining of EBV seen with mAb 104 this antibody was used for these experi- cells. Compared to the other B7-1 antibodies the mAb EW3.4 at ments. PMNs were stimulated with a panel of cytokines. LPS, optimal concentrations showed only weak staining (Fig. 3). IFN- , and IL-12 could induce surface expression of the Staining with mAb 104 in PMNs showed a speckled pattern B7-1-like molecule. This finding was reproduced in six out of and was morphologically localized to the intracytoplasmic ten experiments. Figure 6 shows an example of surface granules. This was determined by the typical size and localiza- staining with mAb 104 on PMNs after stimulation with LPS, tion of the granules on confocal microscopy (Fig. 4). To further IL-12, and IFN- for 24 h. With the other cytokines tested characterize the B7-1-like antigen in PMNs, Western blot was (IL-1, IL-6, G-CSF, GM-CSF, TNF- , IL-8) no surface expres- performed with mAb 104. Several distinct bands were revealed sion of the B7-1-like molecule was observed. that showed a molecular mass between 30 and 65 kDa (Fig. 5) as well as diffuse staining in this region. The molecular mass of Expression of B7 mRNA in neutrophils the observed bands is similar to those described for the B7-1 To test whether B7-1 and B7-2 were present at the transcrip- protein in B cells. tional level we performed RT-PCR for B7-1 and B7-2 mRNA from unstimulated neutrophils and after 6, 10, and 20 h of Surface expression of B7-1-like antigen in PMNs stimulation with different stimuli. Resting PMNs did not after activation express detectable levels of B7-1 or B7-2 mRNA. mRNA Because the B7-1-like molecule is localized to the cytoplasmic expression of B7-1 but not B7-2 was induced in PMNs granules we examined whether it is released from PMNs after stimulated with LPS after 20 h (Fig. 7), whereas after 6 and 10 degranulation. PMNs were stimulated with PMA or fMLP, h of stimulation B7-1 mRNA was not yet detectable. Stimula- which are complete secretagogues. The cell lysates and super- tion with IL-8, IL-12, or IFN- did not induce B7-1 mRNA. natants were collected and examined for the presence of the Other stimuli that did not induce B7-1 mRNA expression were B7-1-like molecule by Western blot. Staining with mAb 104 G-CSF, TNF- , and GM-CSF (not shown). Simultaneously with revealed the same pattern of bands in the cell lysates after PMA B7-1 mRNA expression we observed expression of IL-12 stimulation, whereas in the supernatant there was only weak mRNA in PMNs. staining of a band with a molecular mass of 30 kDa (Fig. 5). The Because B7-1 mRNA was absent from PMNs we investigated same results were obtained with fMLP stimulation (data not whether there was expression of B7-1 in granulopoietic cells in shown). These results indicate that the B7-1-like molecule is the bone marrow. Bone marrow cells were isolated from bone not released by degranulation. marrow aspirate by Ficoll separation and examined by flow Fig. 4. Confocal laser scanning microscopy shows that mAb 104 antigen (a and b) but not B7-2 (c) is expressed in PMNs. High-power optical sections running across the cytoplasm of the PMNs (b) reveal that the epitopes detected are associated with the characteristic small cytoplasmic granules of the PMNs (arrowheads in b), but are absent from the cell surface. Only negligible fluorescence is seen in controls (d) using a nonspecific control mouse IgG1 antibody. a, c, d 330; b 1550. 948 Journal of Leukocyte Biology Volume 66, December 1999 http://www.jleukbio.org
  • 5. Fig. 6. Surface expression of B7-1-like molecule can be induced by stimula- tion of PMNs in vitro. (A) unstimulated, (B) LPS stimulated, (C) IFN- - stimulated, and (E) IL-12-stimulated PMNs; line histogram isotype control, dotted histogram mAb 104 staining. (D) unstimulated PMNs, surface staining with anti-CD16 antibody (dotted histogram) as marker for neutrophils and positive control. (F) PMNs stimulated with IFN- surface staining with B7-2 antibody (dotted histogram). Stimulation with the different cytokines was performed for 24 h. Fig. 5. Detection of B7-1-like molecule in PMNs by Western blot. Whole-cell lysate of unstimulated and PMA-stimulated PMNs as well as supernatant of In PMNs from the CSF of patients with bacterial meningitis as PMA-stimulated PMNs was examined for presence of B7-1-like antigen using well as from peripheral blood of healthy donors, expression of a the mAb104. The molecular mass of the observed bands from cell lysate is in B7-1-like molecule was detected in the cytoplasmic granules. the range of 30 to 65 kDa. After PMA stimulation the B7-1 molecule is still This is a novel finding because expression of costimulatory detected in the cell lysate and only a weak band with a molecular mass of 30 kDa is detected in the supernatant. cytometry. The resulting cell population contained about 30% CD16-positive cells but no mature PMNs. Cells were gated on the CD16 population and intracytoplasmic B7-1 was detected while there was no surface expression of B7-1, similar to the results obtained with PMNs (Fig. 8). Bone marrow cells also expressed B7-1 mRNA as determined by RT-PCR using three sets of B7-1 PCR primers that amplify different regions of the B7-1 cDNA (Fig. 9). It is interesting that with the primers B7-1 and B7-1b PCR product of expected length was obtained, whereas no distinct PCR product could be generated with the primers B7-1a. The B7-1a forward primer is complementary to a sequence encoding the signal peptide, the reverse primer to a sequence in the IgG-like V-type domain. The same pattern was obtained when cDNA from LPS-stimulated PMNs was used (Fig. 9). DISCUSSION Fig. 7. Expression of B7-1 mRNA is induced after stimulation of PMNs with LPS (but not IL-8, IL-12, or IFN- ) for 20 h measured by RT-PCR. We investigated the expression of B7 costimulatory molecules Simultaneously, mRNA of IL-12 p40 is increased, whereas B7-2 mRNA is not in PMNs from inflammatory CSF samples and peripheral blood. detectable. -Actin, 30 PCR cycles; B7-1, B7-2, and IL-12p40, 35 PCR cycles. Windhagen et al. B7-1 expression in human neutrophils 949
  • 6. Fig. 8. Expression of B7-1-like molecule in bone marrow cells. (A) Surface staining with CD16 mAb, (B) surface staining with mAb 104, (C) intracytoplasmic staining with mAb 104. Line histogram isotype control, dotted histograms CD16 or mAb 104. Cells were gated on the CD16- expressing population. molecules in human PMNs has not been reported so far, failure of this antibody to stain PMNs might be due to low although expanding research has been directed toward the sensitivity or might indicate differences in the epitope recog- interaction of the innate and acquired immune systems. nized. Because the B7-1-like molecule in PMNs might represent a Alternatively the B7-1-like molecule may be a distinct novel link between PMNs and T cells, which express two B7 member of the B7 family of costimulatory molecules. A novel ligands, we further characterized this molecule in relation to costimulatory molecule distinct from B7-1 and B7-2 was other B7 molecules. The initial observation was made using the recently described on activated human myoblasts and this mAb 104 that specifically stains B7-1. Four additional B7-1 molecule could be detected by the BB-1 antibody and CTLA4-Ig antibodies were tested and two also recognized B7-1 in PMNs, but not other B7-1 mAbs [25]. This possibility is supported by whereas two did not. There are several possible explanations for our finding that one set of the PCR primers used (B7-1a this B7-1-like staining pattern in PMNs. One hypothesis would primers) does not amplify, whereas the other B7-1 primers be the existence of different glycosylation forms of B7-1 in amplified a PCR product of expected length. The B7-1a primers PMNs similar to findings that were recently described with are complementary to regions in the signal sequence and human B7-2. It was shown that human T cells express a IgG-like V-type domain of the B7-1 cDNA. These findings hypoglycosylated form of B7-2 that has different binding suggest that the B7-1 like molecule in PMNs is different from affinities to CD28 and CTLA4 compared with the B7-2 B7-1 at the mRNA level, possibly at the amino-terminal region expressed on EBV cells [22]. B7-1 has eight potential N-linked containing the IgG-like V-type domain or the signal sequence. glycosylation sites in the extracellular region and the molecular Alternatively, this result could be explained by the existence of mass of the glycosylated protein has been found to be in the different B7-1 splicing forms in PMNs compared with B cells or range of 44–60 kDa, whereas the predicted size of non- monocytes. glycosylated B7-1 is 30 kDa [23]. The B7-1 like molecule in For the PCR analysis it was important to exclude contamina- PMNs shows bands with a molecular mass of between 30 and 65 tion of the PMN preparation with monocytes because these are kDa on Western blot. From this finding it can be concluded that potent producers of B7-1 after stimulation with LPS. For this both molecules are similar in size; the bands at 30 kDa might purpose the PMN preparation was stained with naphthol-AS-D- represent hypo- or non-glycosylated protein. chloroacetate, where no mononuclear cells were detectable. The Ab L307.4, which did not stain the B7-1-like molecule Moreover, monocytes have different kinetics of B7 mRNA in PMNs, recognizes an epitope in the IgG-like V-type domain expression and produce high levels of both B7-1 and B7-2 of B7-1 [24], indicating that there are differences between both mRNA on LPS stimulation with a maximum at about 6 h [26], molecules in this epitope. The epitope recognized by mAb whereas PMNs started B7-1 mRNA expression only after 20 h. EW3.4 is not known. Because the mAb EW3.4 showed only It cannot be ruled out that eosinophils express B7-1 mRNA, weak staining on EBV cells that express high levels of B7-1 the although the amount of B7-1 mRNA detected was not depen- dent on the eosinophil percentage of the PMN preparation. It is interesting that in PMNs stimulated with LPS there was expression of both B7-1 and IL-12 with the same kinetics. Simultaneous expression of B7-1 and IL-12 was previously found in inflammatory lesions from multiple sclerosis brains, indicating that this might be of importance in inflammatory conditions in vivo [20]. Recent findings showed that IL-12 mRNA in neutrophils is expressed after LPS stimulation for 20 h [27]. Other mRNA transcripts are expressed constitutively in peripheral blood PMNs like actin and MHC class I [28]. The absence of B7-1 mRNA in unstimulated PMNs is unusual because in most cell types protein expression in the absence of mRNA expression is not known. However, in PMNs it was Fig. 9. Expression of B7-1 mRNA in bone marrow cells and PMNs using three previously reported that constitutive expression of IL-6 is sets of B7-1 primers spanning different parts of the B7-1 cDNA. B7-1, cDNA present in the cytoplasm in the absence of IL-6 mRNA. In position 499–888, PCR product 389 bp; B7-1a, cDNA position 318–641, PCR product 323 bp; B7-1b, cDNA position 512–1182, PCR product 671 bp. In immature cells in the bone marrow, however, IL-6 mRNA was bone marrow cells and LPS-stimulated PMNs a PCR product is generated with detectable and correlated to the intracytoplasmic level of primers B7-1 and B7-1b, but not B7-1a. circulating neutrophils [29]. Similarly, our results show B7-1 950 Journal of Leukocyte Biology Volume 66, December 1999 http://www.jleukbio.org
  • 7. mRNA expression in bone marrow cells and expression of relevant in inflammatory conditions in vivo. Because surface B7-1-like protein in the CD16 bone marrow population. This expression of the B7-1-like molecule was only detected in the suggests that intracytoplasmic stores of B7-1-like molecule subacute phase of bacterial meningitis it can be hypothesized accumulate during granulopoiesis in the bone marrow. After that other cells possibly in combination with bacterial factors stimulation with LPS mRNA for B7-1 is transcribed, indicating like LPS are important to induce the translocation of B7-1-like that mature PMNs can produce new B7-1-like molecule if their molecule to the cell surface. Alternatively, surface expression survival is prolonged as in inflammatory conditions. of B7-1-like molecule might reflect age or maturation of PMNs B7-1 is known as a surface molecule of APCs that is in the course of chronic inflammation. The possibility that the important for the costimulation of T cells. PMNs have so far not B7-1-like molecule is shed or released from other cells and been associated with T cell costimulation, although they do secondarily bound to PMNs cannot be ruled out, however, this secrete cytokines that can influence T cell function [3]. The would be more likely if all PMNs in the CSF had been positive. expression of B7-1 on APCs requires external stimuli and is After in vitro stimulation of PM a low level of surface expression tightly regulated. B7-1 appears on monocytes and B cells about of B7-1-like molecule could be induced as detected by a shift of 2 days after stimulation and is therefore thought to influence the fluorescence intensity in the whole-cell population. From this it later stages of an immune response [30, 31]. Because the cannot be determined whether all PMNs translocate a small B7-1-like molecule was localized to the intracytoplasmic amount of B7-1-like molecule to the surface or whether this granules we used degranulating stimuli to induce release of phenomenon is a result of B7-1-like molecule released from a B7-1-like molecule from the cells. However, after PMA or fMLP few cells. Because the stimuli used did not induce cell death as stimulation B7-1-like molecule could still be detected in the determined by trypan blue exclusion or loss of B7-1-like cell lysates but not in the cell supernatant in Western blots, molecule in a subpopulation as determined by immunocytochem- indicating that it is not released by degranulation. The faint istry it seems more likely that the whole PMN population band with a molecular mass of 30 kDa that was detectable in translocates B7-1-like molecule from the intracytoplasmic pool the supernatant might represent non-glycosylated protein re- to the cell surface. It is possible that the stimuli used here are leased from small numbers of dying cells. not optimal and that other unknown factors, possibly contact In PMNs intracellular proteins may translocate to the cell with other cells like mononuclear cells at an inflammatory site, surface, e.g., Cd11b and CD45 translocate from granules to the are necessary to induce a stronger surface expression. In the course of an inflammatory reaction PMNs are among cell surface after activation [32] and rapid surface expression the first cells to enter the tissue. Here they can perform a from an intracellular pool was described for molecules of the variety of biological functions, including phagocytosis, cytokine CD18 complex [33]. Expression of B7-1-like molecule on secretion, degranulation, or translocation of stored molecules to PMNs was observed after considerably longer stimulation. the cell surface. PMNs as terminally differentiated cells are relatively short- We have presented evidence for a B7-1-like molecule as a lived, undergoing death by apoptosis. Apoptosis in the mature new member of the B7 family in PMNs from inflammatory CSF neutrophil is a constitutive process that results in a rapid specimen and peripheral blood. This molecule may have turnover of the circulating neutrophil population with a t1/2 of functional activity because surface expression can be induced 5–6 h in vivo [34, 35]. However, survival of neutrophils can be in vitro and was also observed in vivo in an inflammatory CSF greatly extended after exposure to microenvironmental signals specimen. Moreover, the B7-1-like molecule binds the func- involved in infection and immunity such as LPS, streptococci, tional B7 ligand CTLA4-Ig and may therefore have a role in the or several cytokines like IL-6, G-CSF, or GM-CSF [3]. To interaction between PMNs and T cells. determine viability of the neutrophils we performed trypan blue exclusion and CD16 staining [36] and consistently 90% of cells were viable in our experiments. Therefore surface expres- sion of a B7-1-like molecule on PMNs may be of biological ACKNOWLEDGMENTS significance when under optimal in vivo conditions survival is prolonged, particularly at sites of inflammation. Similar to our This work was supported by an internal grant of the Medical findings with the B7-1-like molecule it was reported that PMNs School Hannover (A. W.), and by the Deutsche Forschungsge- may express MHC II on the cell surface after prolonged in vitro meinschaft (Ge 647/2-2 to A. G.). We thank Reinhold Schmidt, culturing [4]. 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