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


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Halaas, O.
Right arrow Articles by Espevik, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Halaas, O.
Right arrow Articles by Espevik, T.
The Journal of Immunology, 1998, 160: 4330-4336.
Copyright © 1998 by The American Association of Immunologists

Induction of Fas Ligand in Murine Bone Marrow NK Cells by Bacterial Polysaccharides1

Øyvind Halaas2, Randi Vik and Terje Espevik

Institute of Cancer Research and Molecular Biology, The Norwegian University of Science and Technology, Trondheim, Norway


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bacterial polysaccharides have a wide range of activities in mammals. We have studied the effect of LPS and poly-ß-(1->4)-D-mannuronate (mannuronan, poly-M), an exopolysaccharide from Pseudomonas aeruginosa, on the cytotoxicity mediated by murine bone marrow cells (BMC). Addition of LPS or mannuronan to BMC induced a time- and dose-dependent cytotoxicity against Jurkat cells. The LPS- or mannuronan-induced cytotoxicity was due to increased Fas ligand (FasL) expression by BMC, since 1) Fas-transfected L1210-Fas target cells were more susceptible to lysis than the Faslow-expressing parent L1210 cells, 2) stimulated BMC from FasL-defective gld/gld mice were not cytolytic and, 3) the cytolytic activity of normal BMC was inhibited by a Fas-Fc fusion protein. Flow cytometry showed an increase in surface FasL in LPS-stimulated BMC. RT-PCR analysis of BMC revealed constitutive expression of FasL mRNA, which was increased after LPS stimulation. Immunomagnetic depletion of NK1.1-, CD2-, or CD32/16-expressing cells from BMC abrogated the LPS-induced BMC cytotoxicity against L1210-Fas cells, suggesting that NK cells were the cytotoxic effector cells. Depletion of CD45R/B220-, Gr-1-, or CD11b/Mac-1-expressing cells only partially decreased BMC-mediated cytotoxicity, and depletion of CD4- or CD8a-expressing cells had no effect. The results support the conclusion that LPS and mannuronan induce expression of cytotoxic FasL on bone marrow NK cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacterial polysaccharides have pleiotropic effects on the immune system, ranging from the beneficial alert of onset of an infection to the lethality of an endotoxic shock. Several organs and tissues are affected by LPS, especially the thymus, liver, and endothelium (1, 2). In sepsis or endotoxemia, a high degree of apoptosis is seen in thymocytes and liver cells, whereas less apoptosis occurs in spleen cells and bone marrow cells (BMC)3 (3, 4, 5). In vitro, LPS causes apoptosis in endothelial cells in synergy with heat shock inducers (6) but prevents neutrophils and monocytes/macrophages from undergoing apoptosis (7, 8, 9). The molecular mechanisms of LPS-induced apoptosis or prevention of apoptosis are not currently known.

The Fas (CD95, Apo-1) and Fas ligand (FasL, CD95L) apoptotic system is well established in regulation of immune cell development and selection as well as in immune responses. In hemopoietic cells, Fas is expressed on monocytes/macrophages (9, 10, 11), granulocytes (10, 11), T and B lymphocytes (12), NK cells (13), and hemopoietic progenitors (14, 15, 16). However, expression of Fas is not always sufficient to make cells susceptible to Fas-mediated apoptosis (10, 11, 17). The FasL effector molecule, originally thought to be expressed only in T cells, has now been shown to be widely expressed by hemopoietic cells. FasL is expressed on T cells (predominantly in Th0 and Th1 CD4+ helper cells and in CD8+ cytotoxic cells) (18), B cells (19), granulocytes (11), NK cells (13, 20), and macrophages (9). FasL is also abundantly expressed in nonhemopoietic immune-privileged tissues and in tissues with a high level of cell turnover (21). Northern blot studies have shown that only very low levels of FasL mRNA are constitutively expressed in murine bone marrow, and bone marrow-derived stromal cell lines are FasL negative (18). However, little is known about the regulation of FasL expression in the bone marrow or the role of Fas-FasL interactions in endotoxemia.

LPS is composed of a polysaccharide attached to a water-insoluble micelle-forming lipid moiety, while poly-ß-(1->4)-D-mannuronate (mannuronan, poly-M) isolated from Pseudomonas aeruginosa is a well-defined polysaccharide with cytokine-inducing activity (22). Both LPS and mannuronan induce the production of TNF, IL-1, and IL-6 from human monocytes (22, 23), bind to membrane CD14 (24), and require either serum- or LPS-binding protein for TNF production at low concentrations (24, 25). While LPS can stimulate CD14-negative cells, mannuronan requires membrane CD14 for biologic activity (24). As opposed to LPS, mannuronan is not toxic in mice even at high concentrations (26). Mannuronan induces colony-stimulating activity from primary bone marrow stroma and is a synergistic factor in murine progenitor cell colony formation (27). Prophylactic administration of mannuronan rescues mice both from lethal irradiation (27) and lethal infection (Ref. 26 and T. Espevik, unpublished results) suggesting interesting therapeutic uses of this polymer.

In this report, we show that both LPS and a defined polymer such as mannuronan stimulate murine bone marrow NK cells to express FasL. Our results may contribute to the understanding of the molecular mechanisms of sepsis and LPS-associated graft-vs-host disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Medium and chemicals

Unless otherwise stated, medium refers to RPMI 1640 (Life Technologies, Paisley, Scotland) with 0.1 mg/ml L-glutamine and 20 µg/ml gentamicin (Garamycin, Schering-Plough, Kenilworth, NJ). Complete medium contained 10% heat-inactivated (30 min, 56°C) FCS (Life Technologies).

Mannuronan was purified from P. aeruginosa as described elsewhere (25). LPS contamination was <10 ng/mg mannuronan, and no protein contamination could be detected in 1 mg mannuronan by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). LPS (Escherichia coli O26:B6) was purchased from Sigma (St. Louis, MO).

A fusion protein with murine Fas-linked to the constant and hinge region of human IgG1 (Fas-Fc) was purified from medium of Fas-Fc-secreting NIH-3T3 cells (kindly provided by Dr. P. Leder, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA) on a protein A column as described elsewhere (17).

Cytokines used were recombinant human (rh)IL-6 (Sandoz, Basel, Switzerland), rhIL-1{alpha} (Glaxo, Geneva, Switzerland), murine rTNF (Genentech, South San Francisco, CA) and rhIL-2 (Cetus Oncology, Emeryville, CA).

Cells and cell lines

BMC flushed from the femora and tibia of 8- to 12-wk-old C57BL/6 mice (Bomholt Gård, Ry, Denmark) were used in all experiments except where indicated. C3H/HeNBom Fas/gld mice (gld) homozygous for nonfunctional mutated FasL (28) were purchased from Bomholt Gård for negative control. L1210 (subclone L1210-3) and Fas-transfected L1210-Fas cells expressing low and high levels of membrane Fas (29, 30), respectively, were generously provided by Dr. Pierre Golstein, INSERM, Marseille, France. d12S-P60C hybridoma (d12S) cells (provided by Dr. P. Golstein) stimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml) (both from Sigma) were used as FasL-positive effector cells (29, 30). Jurkat T cells, which are sensitive to FasL, and the murine RAW macrophage cell line were purchased from American Type Culture Collection (Rockville, MD).

In some experiments, BMC were depleted of specific lineage marker cells by incubating 1 x 108 BMC/ml with 10 µg/ml of rat IgG anti-mouse mAbs (CD2, CD4, CD8a, CD11b/Mac-1, CD45R/B220, Gr-1, or CD32/16; PharMingen, San Diego, CA) or mouse IgG2a anti-mouse NK1.1 (PharMingen) on ice for 30 min, washing three times in medium, and incubating with sheep anti-rat IgG (or rat anti-mouse IgG2a in the case of NK1.1)-coated magnetic Dynabeads (M-450, Dynal, Oslo, Norway) at a bead-to-cell ratio of 10:1 at approximately 5 x 107 cells/ml with gentle rotation for 45 min at 4°C. Flow cytometric analysis showed that 91 to 98% of the cells expressing each lineage marker were depleted by using this method. Lineage-depleted cells (Lin- cells) were prepared using a mixture of the above-mentioned rat Abs (except CD32/16) and a rat anti-erythroid mAb (TER-119; PharMingen) and employing two rounds of depletion using sheep anti-rat magnetic Dynabeads at bead-to-initial cell number ratios of 2:1. The yield of Lin- cells was 2 to 5% of the BMC.

Bone marrow-derived macrophages were obtained by culturing nonadherent BMC in {alpha}-MEM (Life Technologies) supplemented with 0.3 mg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), 10% conditioned medium from L929 cells (source of CSF-1) (31), and 10% heat-inactivated FCS (HyClone Laboratories, Logan, UT) for 1 wk at 37°C.

Primary stromal cell cultures were obtained by seeding 2 x 107 BMC in 10 ml complete {alpha}-MEM with 20% horse serum (BioWhittaker, Walkersville, MD), 20 µg/ml amphotericin B (Fungizone; Bristol-Myers Squibb, New York, NY; complete medium with amphotericin B), and 10 µM hydrocortisone (Sigma) in 25-cm2 flasks for 6 wk at 33°C with weekly semidepopulation of nonadherent cells, then refeeding with 5 ml of complete medium with 20% horse serum and amphotericin B (without hydrocortisone).

Cytotoxicity assay

Chromium release cytotoxicity assays were performed essentially as described elsewhere (17, 29). Briefly, 1 x 107 L1210 or L1210-Fas cells/ml were labeled for 2 h with 18.5 MBq/ml (500 µCi/ml), and 6 x 106 Jurkat cells/ml were labeled for 1.5 h with 22.2 MBq/ml (600 µCi/ml) with sodium [51Cr]chromate (sp. act., 18.5 GBq/mg (500 mCi/mg); DuPont NEN, Boston, MA). The cells were washed three times in medium before 1 x 104 target cells/well were added to effector cells with or without additions in a total volume of 200 µl in round-bottom 96-well plates. To ensure cell:cell contact, the plates were centrifuged at 200 x g for 2 min before the incubation was started. Percentage of specific lysis was calculated from 100% x (experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm), where spontaneous cpm represents radioactivity released from target cells only, and maximum cpm represents radioactivity released from target cells lysed with 0.25% SDS. In some experiments, Fas-Fc fusion protein, anti-TNF mAb (TN3 19.12, 50–100 µg/ml; CellTech, Slough, U.K.), anti-IFN-{gamma} mAb (R4-GA2, 50 µg/ml; PharMingen), anti-IL-6 mAb (20 µg/ml; PharMingen), or anti-IFN-{alpha} mAb (20 µg/ml; HyCult Biotechnology, Uden, The Netherlands) were used for blocking of FasL, TNF, IFN-{gamma}, IL-6, and IFN-{alpha} activities, respectively. Human IgG1 (Sigma) or rat IgG (Pierce, Rockford, IL) were used as controls. Nonspecific binding of Fas-Fc to BMC was blocked with murine IgG (100 µg/ml; Caltag, So. San Francisco, CA). Control Abs alone or in combination with murine IgG did not affect specific lysis of L1210, L1210-Fas, or Jurkat cells. In some experiments, contact between 1 x 105 Jurkat target cells and 3 x 106 BM effector cells was prevented by a 40-µm mesh Transwell (Life Technologies), and the viability of Jurkat cells was assessed by fluorescence microscopy after staining with acridin orange (yielding green fluorescence in viable cells) and ethidium bromide (red fluorescence in dead cells with damaged cell membranes). Jurkat cells cultured without effector cells showed >95% viability throughout these experiments.

Flow cytometry

All steps were done at 0 to 4°C. For detection of surface FasL, cells were preincubated with murine IgG (100 µg/ml) in PBS + 0.1% BSA (Sigma) for 30 min to reduce nonspecific binding before 50 µg/ml mouse IgG2b anti-mouse FasL mAb Kay-10 (PharMingen) was added for 30 min. Bound FasL Abs were detected with 20 µg/ml biotinylated anti-mouse IgG2b (PharMingen) and streptavidin-phycoerythrin (Becton Dickinson, Mountain View, CA). Cells incubated with mouse IgG and subjected to staining in the second and third step served as controls. The cells were analyzed on a FACScan cytometer (Becton Dickinson). Surface FasL was also detected by staining with Fas-Fc (50 µg/ml) for 30 min using FITC-conjugated mouse anti-human IgG1 (20 µg/ml; Zymed Laboratories, San Francisco, CA) as a second step.

Reverse transcriptase-PCR

RNA was extracted from 107 BMC with TRIzol reagent (Life Technologies) as described by the manufacturer. cDNA was synthesized from 2 µg RNA using M-MLV reverse transcriptase (Life Technologies) as previously described (13). PCR was performed on a 1:2 dilution of the cDNA as described (13). Each PCR cycle consisted of a denaturation step at 94°C for 1 min, annealing for 2 min (at a primer-specific temperature), polymerization at 72°C for 1 min, and after completion of the cycles, one 7-min polymerization at 72°C. Primers were (5' to 3'): murine ß-actin 3'-end, GAT CTT GAT CTT CAT GGT GCT AGG; 5'-end, TTG TTA CCA ACT GGG ACG ACA TGG (annealing at 67°C, 20 cycles); murine FasL 3'-end, CCA CCG CCA TCA CAA CC; 5'-end, GGG GCT GGT TGT TGC AA (annealing at 67°C, 28–30 cycles); murine Fas 3'-end, AAC TGC ACC CTG ACC CA; 5'-end, CAG CAC AGG AGC AGC TG (annealing at 69°C, 30 cycles); all primers were synthesized at Kebo Lab, Spånga, Sweden. PCR products were dissolved on a 1.8% (w/v) agarose gel, stained with ethidium bromide, and analyzed on a GelDoc 1000 with Molecular Analyst software (Bio-Rad Laboratories).

Cytokine analysis

TNF was determined by its cytotoxic effects on the fibrosarcoma cell line WEHI 164 clone 13 measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (32) as previously described (33) using murine rTNF as standard. IL-1 was determined using a two-step bioassay employing the murine thymocyte cell line EL-4 NOB-1, which responds to IL-1 with IL-2 production as previously described (34). rhIL-1 was included as standard. IL-2 from IL-1-stimulated NOB-1 cells or from LPS-stimulated BMC was determined in an MTT assay using the IL-2-dependent murine T cell line HT-2 as described (35). IL-6 was determined in an MTT assay using the IL-6-dependent mouse hybridoma cell line B.13.29 clone 9 as described previously (36). rhIL-6 was included as a standard.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time- and dose-dependent induction of BMC-mediated cytotoxicity by LPS and mannuronan

We analyzed the induction of cytotoxicity against the human T cell line Jurkat by murine BMC stimulated with LPS and mannuronan. Addition of LPS (100 ng/ml) or mannuronan (100 µg/ml) to cocultures of BMC and 51Cr-labeled Jurkat cells at an E:T ratio 100:1 resulted in a time-dependent increase in the lysis of Jurkat cells reaching 50 and 60%, respectively, at 24 h. Unstimulated BMC did not induce lysis of Jurkat cells. LPS or mannuronan did not affect the spontaneous lysis of Jurkat cells, showing that BMC were required for lysis of Jurkat cells. Both LPS and mannuronan prompted a dose-dependent increase in BMC-mediated cytotoxicity, which reached a plateau level for lysis of Jurkat cells at 100 ng/ml LPS and 50 µg/ml mannuronan.

When cell:cell contact between BMC and Jurkat cells was prevented by a 0.4-µm mesh Transwell physical barrier, Jurkat cells were not killed after 48 h in coculture with BMC in the presence of LPS (100 ng/ml). Hence, LPS-induced BMC lysis of Jurkat cells is cell mediated and requires physical contact between the effector and target cells.

Involvement of FasL in LPS and mannuronan induced BMC cytotoxicity

We hypothesized that FasL was responsible for the cytotoxic activity, since Jurkat cells are sensitive to FasL but not to TNF (data not shown). The involvement of FasL was analyzed by employing Fas-transfected L1210-Fas cells and Faslow L1210 parent cells as target cells in 51Cr release assays (29, 30). L1210-Fas cells were more susceptible than L1210 cells to lysis by stimulated BMC, suggesting that the main cytotoxic effector molecule is FasL (Fig. 1Go). LPS or mannuronan did not affect the spontaneous lysis of L1210 or L1210-Fas cells. Specific lysis was somewhat lower for L1210-Fas cells compared with Jurkat cells, but both time and dose dependency for LPS and mannuronan were similar. The specific lysis of L1210-Fas cells by the FasL+ d12S hybridoma at an E:T ratio 3:1 was 80%, while specific lysis of L1210 cells was 20%, suggesting that L1210 cells expressed low levels of Fas. Also, flow cytometric analysis showed a low level of expression of Fas by L1210 cells. Expression of Fas by L1210 cells has also been shown by others (30, 37). Hence, the presence of Fas on the L1210 parent cell line may account for the susceptibility to lysis by BMC stimulated with LPS or mannuronan (Fig. 1Go).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 1. Involvement of FasL in LPS- or mannuronan-induced BMC cytotoxicity. The specific lysis of L1210 cells (low Fas expression; upper panel) or L1210-Fas cells (high Fas expression; lower panel) by BMC after 22 h of coculture in the absence (ctrl) or presence of LPS (100 ng/ml) or mannuronan (50 µg/ml) at different E:T ratios. The results are mean ± SEM of 2 to 5 independent experiments in triplicates. *p < 0.05, **p < 0.005, and ***p < 0.001 by paired Student’s t test.

 
C3H/HeN gld/gld mice have a point mutation in the FASL gene resulting in a defective FasL protein that is unable to bind to and signal through Fas (28). BMC from C3H/HeN gld/gld mice were not able to lyse L1210, L1210-Fas, or Jurkat cells in the presence of LPS (Fig. 2Go), confirming that FasL is the major cytotoxic molecule in LPS-stimulated BMC.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 2. LPS-stimulated BMC from FasL-defective C3H/HeN gld/gld mice do not mediate cytotoxicity. The specific lysis of L1210 (left), L1210-Fas (middle), and Jurkat cells (right) (all 104 cells/well) by BMC from normal C57BL or FasL-defective C3H/HeN gld/gld mice after 22 h of coculture in the presence of LPS (100 ng/ml) at different E:T ratios. The results are the mean ± SEM of two to four independent experiments in triplicates. *p < 0.05, **p < 0.005, and ***p < 0.001 by Student’s t test.

 
In addition, BMC-mediated cytotoxicity was inhibited by a Fas-Fc fusion protein. The inhibition of cytolysis of L1210-Fas cells with Fas-Fc (100 µg/ml) was 64 and 47% for mannuronan and 57 and 61% for LPS at E:T ratios of 50:1 and 100:1, respectively (Fig. 3Go). Also, lysis of Jurkat cells by LPS-stimulated BMC was inhibited 40% by Fas-Fc (60 µg/ml) at an E:T ratio of 100:1 (not shown). The blocking anti-FasL mAb Kay-10 (20 µg/ml) also inhibited the specific lysis of L1210-Fas cells 50% when LPS-stimulated BMC at an E:T ratio of 50:1 were used as effector cells (not shown). Fas-Fc (10 µg/ml and 50 µg/ml) completely inhibited the cytolytic activity of FasL+ d12S cells against L1210-Fas in short term 51Cr release assays (4 h), but only by 55 to 60% in long term assays (24 h).



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 3. Inhibition of BMC-mediated cytotoxicity by a Fas-Fc fusion protein. The specific lysis of L1210-Fas cells cocultured with BMC for 22 h with LPS (100 ng/ml) or mannuronan (50 µg/ml) in the presence or absence of Fas-Fc fusion protein (100 µg/ml). Cells were preincubated with murine IgG (100 µg/ml) to reduce unspecific binding. Addition of isotype-matched human IgG1 control did not affect specific lysis (-Fas-Fc in figure). Results are mean ± SD of triplicate determinations from one representative experiment repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 by paired Student’s t test.

 
These experiments provide evidence that BMC stimulated with LPS or mannuronan express FasL, which is a major cytotoxic effector molecule against L1210-Fas and Jurkat cells.

Induction of surface FasL and FasL mRNA in murine BMC by LPS

The expression of FasL was further analyzed by flow cytometry and RT-PCR. Adding LPS to unfractionated BMC for 24 h resulted in a marked increase in the expression of surface FasL (Fig. 4Go). The histogram shown in Figure 4GoB suggests that FasL is expressed mainly by a subpopulation of LPS-stimulated BMC. Only low levels of surface FasL were detected on freshly isolated BMC (data not shown). Similar results were obtained when Fas-Fc was used to detect surface FasL.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 4. Expression of surface FasL on BMC. Representative histograms of FasL expression in BMC cultured for 24 h without (A) or with LPS (100 ng/ml) (B). The cells were stained for FasL using mouse IgG2b anti-mouse FasL mAb Kay-10, biotinylated anti-mouse IgG2b, and streptavidin-phycoerythrin. Cells were preincubated with mouse IgG to reduce nonspecific binding. Cells incubated with mouse IgG and stained with the second and third step served as control.

 
RT-PCR showed that the FASL gene was constitutively transcribed in BMC (Fig. 5Go). FasL mRNA levels increased in BMC stimulated with LPS for 6 and 12 h, but after 24 h with LPS, the FasL mRNA level was reduced to that of freshly isolated BMC. BMC incubated in medium alone retained the FasL mRNA level until a drop after 24 h of culture (Fig. 5Go). Fas, however, was not regulated at the transcriptional level (data not shown). The data show that both transcription of the FASL gene and induction of surface FasL occur in LPS-stimulated BMC, which is in agreement with the bioassays.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 5. RT-PCR analysis of FasL transcription in BMC. RT-PCR was performed on 2 µg of total RNA from BMC and analyzed using a GelDoc 1000. Shown are ß-actin (20 PCR cycles) and FasL (28 cycles) mRNA levels in BMC after 0, 6, 12, and 24 h in culture without (-) or with (+) LPS (100 ng/ml) from one representative experiment repeated three times.

 
Involvement of bone marrow NK cells in LPS-stimulated BMC-mediated cytotoxicity

We next characterized the cell type responsible for BMC-mediated cytotoxicity. Macrophages respond readily to LPS, but neither macrophages derived from bone marrow nor murine RAW macrophages had any cytotoxic activity against L1210-Fas or Jurkat cells in the presence of LPS (data not shown). We performed immunomagnetic depletion of lineage specific cells from fresh BMC before initiating the cytotoxicity assays. All of the cytotoxic activity resided in the CD2 population (Fig. 6GoA). In the mouse, CD2 is expressed on T, B, and NK cells, but since depletion of CD4+ or CD8a+ T cells did not affect the specific lysis and depletion of CD45R/B220+ B cells only modestly decreased the lysis of L1210-Fas cells, it seemed likely that NK cells were responsible for the BMC-mediated cytotoxicity. The NK1.1 Ag is considered a specific NK cell marker, and BMC depleted of cells expressing NK1.1 did not mediate any cytotoxicity in the presence of LPS even at high E:T ratios (Fig. 6Go, A and B). CD32/16 (Fc{gamma}RII/III) is expressed in high amounts on NK cells, and BMC depleted of cells expressing CD32/16 also displayed low levels of cytotoxicity (Fig. 6GoA). Depletion of cells expressing Gr-1 (primarily granulocytes) or CD11b/Mac-1 (primarily myeloid cells) resulted in a partial decrease in LPS-stimulated BMC-mediated cytotoxicity (Fig. 6GoA), suggesting that myeloid cells are involved in LPS-induced FasL-mediated cytotoxicity. Lin- cells, composed of stromal cells and hemopoietic progenitors, did not mediate cytotoxicity (Fig. 6GoA). Six-week-old primary stromal culture cells stimulated with LPS or mannuronan also did not mediate cytotoxicity against L1210-Fas cells, although FasL transcripts were found by PCR (data not shown). These data demonstrate that NK cells are the major FasL effector cells in murine BMC-stimulated with LPS.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 6. Involvement of NK cells in LPS-induced BMC cytotoxicity. A, The specific lysis of L1210-Fas cells cocultured with fresh BMC immunomagnetically depleted of lineage marker-expressing cells for 22 h in the presence or absence of LPS (100 ng/ml) at E:T ratio 50:1. Results are mean ± SD of one (Lin-), two (CD4, NK1.1, CD32/16), or three independent experiments in triplicates. B, The specific lysis of L1210-Fas cells cocultured for 22 h with unfractionated or NK1.1+ cell-depleted BMC in the absence or presence of LPS (100 ng/ml) at different E:T ratios. *p < 0.05, **p < 0.005 by paired Student’s t test.

 
IL-1, IL-2, TNF, IL-6, IFN-{alpha}, and IFN-{gamma} are not involved in LPS-induced cytotoxicity

Immunomagnetically separated NK cells responded poorly to LPS in induction of cytotoxicity compared with unseparated BMC, and myeloid cells did not seem to participate in the cytotoxic activity. It is possible, therefore, that LPS indirectly induces NK cell FasL expression through production of endogenous cytokines. LPS induced production of bioactive TNF, IL-1, and IL-6 from BMC. However, rTNF (10 ng/ml), IL-6 (10–100 ng/ml), or IL-1 (10 ng/ml) individually or combined did not significantly modulate BMC cytotoxicity alone or in combination with LPS. Moreover, neutralizing mAbs to TNF (50 µg/ml) or IL-6 (10 µg/ml) did not decrease LPS-induced BMC cytotoxicity. Furthermore, neutralizing mAbs to IFN-{gamma} (50 µg/ml) or IFN-{alpha} (20 µg/ml) did not inhibit LPS-induced BMC cytotoxicity. IL-2, but not IL-7 and IL-12, has been shown to induce FasL expression in human NK cells (13). In agreement with this, IL-2 (at concentrations >4 U/ml) alone or in combination with LPS increased murine BMC-mediated Fas-based cytotoxicity (data not shown). The effect of IL-2 and LPS was additive or less than additive. However, the IL-2 level in BMC cultures (at 5 x 106 BMC/ml, corresponding to an E:T ratio of 100:1) stimulated with LPS for 22 h was below the detection level of the assay (<1 U/ml). An IL-2 concentration of 20 to 100 U/ml was required to obtain a BMC-mediated lysis comparable to 100 ng/ml LPS. Hence, LPS-induced FasL expression in murine BMC is not likely to be mediated by the NK cell-activating cytokine IL-2 nor by the LPS-induced inflammatory cytokines IL-1, IL-6, TNF, IFN-{alpha}, or IFN-{gamma}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have shown that bacterial polysaccharides induce expression of FasL in murine bone marrow NK cells. BMC stimulated with LPS or mannuronan expressed a dose- and time-dependent cytotoxicity. The cytotoxicity was due to increased FasL expression, since 1) Fas-transfected L1210-Fas cells were more susceptible to lysis by LPS- or mannuronan-stimulated BMC than Faslow-expressing L1210 parent cells, 2) LPS-stimulated BMC from FasL-defective gld/gld mice were not cytolytic against L1210-Fas or Jurkat cells, and 3) LPS- or mannuronan-induced BMC cytotoxicity was inhibited by a Fas-Fc fusion protein. In addition, we demonstrated that FasL was up-regulated by LPS at the mRNA and surface protein level.

LPS-induced BMC cytotoxicity was mediated mainly by NK cells, since all cytotoxic activity was removed by depleting BMC of NK1.1+, CD2+, or CD32/16+ cells. Although other cell types present in the bone marrow also express FasL, their contribution seems to be of minor importance. CD4+ and CD8+ T cells, in which FasL was first found (18, 29), did not participate in cytolysis, either because they were not activated or because of their low frequency of occurrence in the bone marrow. Hahne et al. (19) have shown that purified splenic B cells express FasL after stimulation with 50 µg/ml LPS for 3 to 4 days. We observed that BMC stimulated with 100 ng/ml LPS for less than 24 h mediated high cytotoxic activity. Furthermore, depletion of cells with the pan-B marker B220/CD45R from the BMC resulted in only a small and statistically insignificant reduction in the cytolytic activity. Both granulocytes (11) and monocytes/macrophages (9) express FasL, but depletion of Gr-1+ or CD11b+ cells only partially decreased the cytolytic activity. These data suggest that B cells, granulocytes, and macrophages contribute to a minor extent to the FasL activity, but that NK cells are the major cytotoxic cells in LPS-stimulated BMC. Both murine and human NK cells have been shown to express FasL (13, 20), and NK cell cytotoxicity is increased after stimulation with LPS (38). However, the contribution of FasL to LPS-stimulated NK cell cytotoxicity has not previously been demonstrated.

The mechanism by which LPS and mannuronan induce FasL on NK cells in the bone marrow is not known. CD14 is a receptor for both LPS and mannuronan (24). Soluble CD14 can substitute for membrane CD14 in LPS-induced effects on cells lacking membrane CD14; however, mannuronan does not activate cells through a soluble CD14 pathway (24). Thus, the biologic similarities between LPS and mannuronan are restricted to cells expressing membrane CD14. Since NK cells do not express CD14 (39) but are activated by mannuronan, CD14+ BMC may participate in the LPS/mannuronan response by producing cytokines that increase FasL expression in NK cells. As shown in Figure 6GoA, depletion of myeloid CD11b/Mac-1+ and Gr-1+ cells resulted in only a partial and statistically insignificant decrease in cytolytic activity. Since other cells such as fibroblasts and endothelial cells also are able to produce inflammatory cytokines upon LPS stimulation (40), depletion of one cell type can be compensated for by the others. This may explain the low level of decrease in cytolytic activity of BMC depleted of myeloid cells.

LPS and mannuronan induced IL-1, IL-6, and TNF production in murine BMC, but none of these cytokines affected Fas-mediated cytotoxicity individually or in combination. Furthermore, neutralizing mAbs to IL-6, TNF, IFN-{alpha}, and IFN-{gamma} did not modulate LPS-induced cytotoxic activity. It is unlikely, therefore, that these inflammatory cytokines are responsible for the up-regulation of FasL. Although IL-2 alone increased FasL-based cytotoxicity in murine NK cells, LPS-induced cytotoxicity probably does not involve IL-2 for three reasons. First, <1 U/ml of IL-2 was detected in LPS-stimulated BMC cultures, while >4 U/ml of IL-2 was required for a detectable increase in cytotoxicity. Second, the effect of LPS and IL-2 was additive or less than additive even at low doses of IL-2, showing that substantial amounts of IL-2 are required for induction of cytotoxicity also in combination with LPS. Finally, the IL-2 dose equivalent for a 30% specific lysis of L1210-Fas cells (obtained with 100 ng/ml LPS) was 20 to 100 U/ml, while <1 U/ml was detected in LPS-stimulated BMC cultures. Whether other NK-activating cytokines, such as IL-15 (41) or IL-18 (42), participate in LPS-induced expression of FasL has yet to be determined.

The functional consequence of FasL expression on NK cells in the bone marrow may be increased apoptosis of Fas-expressing cells. While lymphoid cells are susceptible to apoptosis in response to LPS or infection, Fas-expressing myeloid cells appear to be rescued by LPS (3, 7, 8, 9, 43). Human bone marrow hemopoietic progenitor cells are sensitive to Fas-mediated apoptosis (14, 15), but whether progenitor and stem cells in the marrow are affected by LPS-induced FasL expression has yet to be studied. Importantly, ligation of Fas does not necessarily lead to apoptosis, since both intracellular proteins (44, 45) and cytokines such as granulocyte-CSF, granulocyte/macrophage-CSF, TNF, and IFN-{gamma} (9, 11) can inhibit Fas-based apoptosis in various cell types. Further studies need to be done to clarify the physiologic significance of LPS-induced expression of FasL in the bone marrow.

The data shown in this report establish for the first time a link between LPS, NK cells, and FasL in the bone marrow. These are central mediators in the pathogenesis of acute graft-vs-host disease (43, 46, 47) and sepsis (1, 2, 3, 4). Hence, our results may contribute to a better understanding of the molecular mechanisms at play in these disorders.


   Footnotes
 
1 This work was supported by the Norwegian Cancer Society, the Norwegian Research Council, and Pronova Biopolymer. Back

2 Address correspondence and reprint requests to Dr. Øyvind Halaas, MSc, Institute of Cancer Research and Molecular Biology, University Medical Center, The Norwegian University of Science and Technology, N-7005 Trondheim, Norway. E-mail address: Back

3 Abbreviations used in this paper: BMC, bone marrow cells; FasL, Fas ligand; gld, generalized lymphoproliferative disease; rh, recombinant human. Back

Received for publication June 18, 1997. Accepted for publication January 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Morrison, D., J. Ulevitch. 1978. The effects of bacterial endotoxins on host mediation systems: a review. Am. J. Pathol. 93:525.
  2. Bone, R. C.. 1991. The pathogenesis of sepsis. Ann. Intern. Med. 115:457.
  3. Ayala, A., C. D. Herdon, D. L. Lehman, C. A. Ayala, I. H. Chaudry. 1996. Differential induction of apoptosis in lymphoid tissues during sepsis: variation in onset, frequency, and the nature of the mediators. Blood 87:4261.[Abstract/Free Full Text]
  4. Zhang, X. M., A. Morikawa, K. Takahashi, G. Z. Jiang, Y. Kato, T. Sugiyama, M. Kawai, M. Fukada, T. Yokochi. 1994. Localization of apoptosis (programmed cell death) in mice by administration of lipopolysaccharide. Microbiol. Immunol. 38:669.[Medline]
  5. Haendeler, J., U. K. Meßmer, B. Brüne, E. Neugebauer, S. Dimmeler. 1996. Endotoxic shock leads to apoptosis in vivo and reduces Bcl-2. Shock 6:405.[Medline]
  6. Buchman, T. G., P. A. Abello, E. H. Smith, G. B. Bulkley. 1993. Induction of heat shock response leads to apoptosis in endothelial cells previously exposed to endotoxin. Am. J. Physiol. 265:H165.[Abstract/Free Full Text]
  7. Colotta, F., F. Re, N. Polentarutti, S. Sozzani, A. Mantovani. 1992. Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 80:2012.[Abstract/Free Full Text]
  8. Mangan, D. F., G. R. Welch, S. M. Wahl. 1991. Lipopolysaccharide, tumor necrosis factor-{alpha}, and IL-1 ß prevent programmed cell death (apoptosis) in human peripheral blood monocytes. J. Immunol. 146:1541.[Abstract]
  9. Kiener, P. A., P. M. Davis, G. C. Starling, C. Mehlin, S. J. Klebanoff, J. A. Ledbetter, W. C. Liles. 1997. Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages. J. Exp. Med. 185:1511.[Abstract/Free Full Text]
  10. Iwai, K., T. Miyawaki, T. Takizawa, A. Konno, K. Ohta, A. Yachie, H. Seki, N. Taniguchi. 1994. Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils. Blood 84:1201.[Abstract/Free Full Text]
  11. Liles, W. C., P. A. Kiener, J. A. Ledbetter, A. Aruffo, S. J. Klebanoff. 1996. Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils. J. Exp. Med. 184:429.[Abstract/Free Full Text]
  12. Nagata, S., P. Golstein. 1995. The Fas death factor. Science 267:1449.[Abstract/Free Full Text]
  13. Medvedev, A. E., A.-C. Johnsen, J. Haux, B. Steinkjer, K. Egeberg, D. H. Lynch, A. Sundan, T. Espevik. 1997. Regulation of Fas and Fas-ligand expression in NK cells by cytokines and the involvement of Fas-ligand in NK/LAK cell-mediated cytotoxicity. Cytokine 9:394.[Medline]
  14. Maciejewski, J., C. Selleri, S. Anderson, N. S. Young. 1995. Fas antigen expression on CD34+ human marrow cells is induced by interferon {gamma} and tumor necrosis factor {alpha} and potentiates cytokine-mediated hematopoietic suppression in vitro. Blood 85:3183.[Abstract/Free Full Text]
  15. Nagafuji, K., T. Shibuya, M. Harada, S. Mizuno, K. Takenaka, T. Miyamoto, T. Okamura, H. Gondo, Y. Niho. 1995. Functional expression of Fas antigen (CD95) on hematopoietic progenitor cells. Blood 86:883.[Abstract/Free Full Text]
  16. Barcena, A., S. W. Park, B. Banapour, M. O. Muench, E. Mechetner. 1996. Expression of Fas/CD95 and Bcl-2 by primitive hematopoietic progenitors freshly isolated from human fetal liver. Blood 88:2013.[Abstract/Free Full Text]
  17. Alderson, M. R., R. J. Armitage, E. Maraskovsky, T. W. Tough, E. Roux, K. Schooley, F. Ramsdell, D. H. Lynch. 1993. Fas transduces activation signals in normal human T lymphocytes. J. Exp. Med. 178:2231.[Abstract/Free Full Text]
  18. Suda, T., T. Okazaki, Y. Naito, T. Yokota, N. Arai, S. Ozaki, K. Nakao, S. Nagata. 1995. Expression of the Fas ligand in cells of T cell lineage. J. Immunol. 154:3806.[Abstract]
  19. Hahne, M., T. Renno, M. Schroeter, M. Irmler, L. French, T. Bornard, H. R. MacDonald, J. Tschopp. 1996. Activated B cells express functional Fas ligand. Eur. J. Immunol. 26:721.[Medline]
  20. Arase, H., N. Arase, T. Saito. 1995. Fas-mediated cytotoxicity by freshly isolated natural killer cells. J. Exp. Med. 181:1235.[Abstract/Free Full Text]
  21. French, L. E., M. Hahne, I. Viard, G. Radlgruber, R. Zanone, K. Becker, C. Muller, J. Tschopp. 1996. Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover. J. Cell Biol. 133:335.[Abstract/Free Full Text]
  22. Otterlei, M., A. Sundan, G. Skjåk-Bræk, L. Ryan, O. Smidsrød, T. Espevik. 1993. Similar mechanisms of action of defined polysaccharides and lipopolysaccharides: characterization of binding and tumor necrosis factor {alpha} induction. Infect. Immun. 61:1917.[Abstract/Free Full Text]
  23. Otterlei, M., K. Ø stgaard, G. Skjåk-Bræk, O. Smidsrød, P. Soon Shiong, T. Espevik. 1991. Induction of cytokine production from human monocytes stimulated with alginate. J. Immunother. 10:286.
  24. Espevik, T., M. Otterlei, G. Skjåk-Bræk, L. Ryan, S. D. Wright, A. Sundan. 1993. The involvement of CD14 in stimulation of cytokine production by uronic acid polymers. Eur. J. Immunol. 23:255.[Medline]
  25. Jahr, T. G., L. Ryan, A. Sundan, H. S. Lichenstein, G. Skjåk-Bræk, T. Espevik. 1997. Induction of tumor necrosis factor production from monocytes stimulated with mannuronic acid polymers and involvement of lipopolysaccharide-binding protein, CD14, and bactericidal/permeability-increasing factor. Infect. Immun. 65:89.[Abstract]
  26. Skaugrud, Ø., J. E. Melvik, M. Dornish, G. Skjåk-Bræk, and T. Espevik. 1997. Prophylaxis of LPS- or pathogen-induced sepsis with poly mannuronic acid (poly M). In IBC’s Seventh Annual International Conference on Sepsis (Abstr.).
  27. Halaas, Ø., W. M. Olsen, O. P. Veiby, D. Løvhaug, G. Skjåk-Bræk, R. Vik, T. Espevik. 1997. Mannuronan enhances survival of lethally irradiated mice and stimulates murine haematopoiesis in vitro. Scand. J. Immunol. 46:358.[Medline]
  28. Hahne, M., M. C. Peitsch, M. Irmler, M. Schroter, B. Lowin, M. Rousseau, C. Bron, T. Renno, L. French, J. Tschopp. 1995. Characterization of the non-functional Fas ligand of gld mice. Int. Immunol. 7:1381.[Abstract/Free Full Text]
  29. Rouvier, E., M. F. Luciani, P. Golstein. 1993. Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity. J. Exp. Med. 177:195.[Abstract/Free Full Text]
  30. Kägi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, H. Hengartner, P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265:528.[Abstract/Free Full Text]
  31. Stanley, E. R.. 1985. The macrophage colony-stimulating factor, CSF-1. Methods Enzymol. 116:564.[Medline]
  32. Mosmann, T.. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65:55.[Medline]
  33. Espevik, T., J. Nissen Meyer. 1986. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immunol. Methods 95:99.[Medline]
  34. Gearing, A. J., C. R. Bird, A. Bristow, S. Poole, R. Thorpe. 1987. A simple sensitive bioassay for interleukin-1 which is unresponsive to 103 U/ml of interleukin-2. J. Immunol. Methods 99:7.[Medline]
  35. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, R. L. Coffman. 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2348.[Abstract]
  36. Aarden, L. A., E. R. De Groot, O. L. Schaap, P. M. Lansdorp. 1987. Production of hybridoma growth factor by human monocytes. Eur. J. Immunol. 17:1411.[Medline]
  37. Yang, Y., M. Mercep, C. F. Ware, J. D. Ashwell. 1995. Fas and activation-induced Fas ligand mediate apoptosis of T cell hybridomas: inhibition of Fas ligand expression by retinoic acid and glucocorticoids. J. Exp. Med. 181:1673.[Abstract/Free Full Text]
  38. Vaillier, D., R. Daculsi, N. Gualde. 1992. Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E2 and interferons. Cancer Immunol. Immunother. 35:395.[Medline]
  39. Griffin, J. D., T. Hercend, R. Beveridge, S. F. Schlossman. 1983. Characterization of an antigen expressed by human natural killer cells. J. Immunol. 130:2947.[Abstract]
  40. Chaby, R., R. Girard. 1993. Interaction of lipopolysaccharides with cells of immunological interest. Eur. Cytokine Netw. 4:399.[Medline]
  41. Carson, W. E., J. G. Giri, M. J. Lindemann, M. L. Linett, M. Ahdieh, R. Paxton, D. Anderson, J. Eisenmann, K. Grabstein, M. A. Caligiuri. 1994. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J. Exp. Med. 180:1395.[Abstract/Free Full Text]
  42. Tsutsui, H., K. Nakanishi, K. Matsui, K. Hagashino, H. Okamura, Y. Miyazawa, K. Kaneda. 1996. IFN-gamma-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J. Immunol. 157:3967.[Abstract]
  43. Baker, M. B., R. L. Riley, E. R. Podack, R. B. Levy. 1997. Graft-versus-host disease-associated lymphoid hypoplasia and B cell dysfunction is dependent upon donor T cell-mediated Fas-ligand function, but not perforin function. Proc. Natl. Acad. Sci. USA 94:1366.[Abstract/Free Full Text]
  44. Okura, T., L. Gong, T. Kamitani, T. Wada, I. Okura, C. F. Wei, H. M. Chang, E. T. Yeh. 1996. Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J. Immunol. 157:4277.[Abstract]
  45. Sato, T., S. Irie, S. Kitada, J. C. Reed. 1995. FAP-1: a protein tyrosine phosphatase that associates with Fas. Science 268:411.[Abstract/Free Full Text]
  46. Nestel, F. P., K. S. Price, T. A. Seemayer, W. S. Lapp. 1992. Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor {alpha} during graft-versus-host disease. J. Exp. Med. 175:405.[Abstract/Free Full Text]
  47. MacDonald, G. C., J. G. Gartner. 1992. Prevention of acute lethal graft-versus-host disease in F1 hybrid mice by pretreatment of the graft with anti-NK-1.1 and complement. Transplantation 54:147.[Medline]



This article has been cited by other articles:


Home page
 CVIHome page
A. Ruiz-Bravo, M. Jimenez-Valera, E. Moreno, V. Guerra, and A. Ramos-Cormenzana
Biological Response Modifier Activity of an Exopolysaccharide from Paenibacillus jamilae CP-7
Clin. Vaccine Immunol., July 1, 2001; 8(4): 706 - 710.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
T. H. Flo, O. Halaas, S. Torp, L. Ryan, E. Lien, B. Dybdahl, A. Sundan, and T. Espevik
Differential expression of Toll-like receptor 2 in human cells
J. Leukoc. Biol., March 1, 2001; 69(3): 474 - 481.
[Abstract] [Full Text]

Home page
 ScienceHome page
H. Grassmé, S. Kirschnek, J. Riethmueller, A. Riehle, G. von Kürthy, F. Lang, M. Weller, and E. Gulbins
CD95/CD95 Ligand Interactions on Epithelial Cells in Host Defense to Pseudomonas aeruginosa
Science, October 20, 2000; 290(5491): 527 - 530.
[Abstract] [Full Text]

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

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS