|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on November 21, 2002; DOI 10.1182/blood-2002-07-2261.
PHAGOCYTES
From the University Medical Hospital, Department
Hematology/Oncology, University of Tübingen, Tübingen,
Germany; and the Institute for Cell Biology, Department
Immunology, University of Tübingen, Tübingen,
Germany.
The endoplasmic reticulum (ER)-resident heat
shock protein Gp96 is involved in protein folding and is
released into the extracellular space after necrotic cell death. In
this context, Gp96 has immunostimulatory properties: it activates
dendritic cells or macrophages and delivers associated peptides into
the antigen presentation pathway, resulting in the induction of
specific T-cell responses. The inflammatory response after necrotic
tissue damage leads to the recruitment of polymorphonuclear neutrophils
(PMNs) and monocytes, allowing them to make their first encounter with
Gp96. We therefore investigated whether PMNs and monocytes interact
with Gp96. We were able to show that PMNs and monocytes specifically
bind fluorescein isothiocyanate (FITC)-conjugated Gp96. The
binding of Gp96-FITC was competed by lipopolysaccharide (LPS) or
fucoidan, a known inhibitor of scavenger receptors. Interestingly, the
binding of LPS-FITC was also competed not only by fucoidan, but by
Gp96, suggesting that LPS and Gp96 share a common receptor on PMNs. One
important effector function of PMNs is the clearance of an inflammatory
site by phagocytosis. We therefore assessed the influence of Gp96 on
phagocytic activity using fluorochrome-labeled polystyrene beads. We
found a marked enhancement of phagocytosis in the presence of Gp96 and
concluded that PMNs not only bind Gp96, but are also activated by
it. Additionally, Gp96-stimulated PMNs and especially monocytes
release large amounts of interleukin-8, a potent neutrophil-attracting
chemokine. In conclusion, we demonstrate that Gp96 specifically binds
to and activates PMNs and monocytes, extending the function of Gp96 as a danger signal to additional members of the innate immune system.
(Blood. 2003;101:2810-2815) The heat shock protein (HSP) Gp96 is localized in
the endoplasmic reticulum (ER) and has a variety of roles in mammalian
organisms. As a chaperone, it is involved in protein folding to prevent
aggregation of partially unfolded proteins in the ER.1
Gp96 also associates with intracellular peptides representative of the
cellular protein content and is able to shuttle these peptides into the
major histocompatibility (MHC) class I presentation
pathway.2-4
After severe tissue damage, necrotic cell death induced by freeze-thaw
cycles,5 or virus infection,6 Gp96 is
released into the extracellular space where it reveals its
immunostimulatory effects: Gp96 preparations from tumor cells have been
shown to elicit protective and therapeutic responses against the tumor from which the HSP had been purified.7,8 The specificity of this immune response is attributed to tumor-derived peptides associated with Gp96.7 After receptor-mediated endocytosis of Gp96 by professional antigen-presenting cells (APCs),9
these peptides are presented on MHC class I molecules,2,3
a process usually called cross-presentation10,11 and one
which might represent a key event during the priming of naive T
cells.12 In addition, Gp96 mediates APC activation that
results in the up-regulation of costimulatory molecules and the release
of the proinflammatory and TH1-promoting cytokines, tumor
necrosis factor Receptor-mediated interactions with Gp96 have been demonstrated for
only professional APCs, including dendritic cells (DCs), macrophages,
and B cells. In addition to these cells, we were recently able to show
that activated platelets, in particular, also specifically bind Gp96
resulting in the reduced activation of DCs.19
However, additional cell types can be expected to make contact with
Gp96. Necrotic cell death after virus infection or severe tissue damage
is followed by inflammatory reactions resulting in the recruitment of
cells from the blood. The majority of these cells are not professional
APCs, such as DCs or macrophages, but polymorphonuclear neutrophils
(PMNs) and monocytes.20 These cells will make contact with
Gp96 because of the fact that necrotic cell death results in the
release of HSPs.5,6 PMNs and monocytes play an important
part in the first line of defense against microbial pathogens by
contributing substantially to the innate host defense with their
ability to rapidly extravasate into inflamed tissue and their use of
potent effector mechanisms (ie, phagocytosis; production of
reactive oxygen species; and release of mediators and antimicrobial
substances).21-23
By taking the immunostimulatory capacity of Gp96 and the role of PMNs
and monocytes as potent effector cells of innate host defense
mechanisms into consideration, we assessed whether these cells may also
be able to interact with Gp96. Here we present for the first time
evidence that PMNs and monocytes are able to specifically bind Gp96 and
respond to Gp96 contact by up-regulation of effector functions.
Materials
Purification of cells
Stimulation of cells Purified PMNs or MNCs were cultivated in RPMI 1640 (PAN Biotech, Aidenbach, Germany) with 3% (vol/vol) heat-inactivated (30 minutes at 56°C) fetal bovine serum (PAN Biotech) and various stimuli as indicated. Cells were suspended in a concentration of 2 × 106 cells/mL and cultivated at 37°C with 7.5% CO2 in air.Antibodies and stainings for flow cytometry The following antibodies were used for analysis by fluorescence-activated cell sorter (FACS): FITC-labeled monoclonal antibodies (mAbs) against CD14 (BD Pharmingen, Hamburg, Germany) or CD66b (Coulter Immunotech, Hamburg, Germany); R-phycoerythrin (PE)-labeled mAbs against CD11b, CD91, CD36, and IL-8 (all purchased from BD Pharmingen). For FACS analysis, aliquots of 2 × 105 cells were incubated with labeled antibodies or protein in (FACS buffer phosphate-buffered saline [PBS] supplemented with 1% [wt/vol] bovine serum albumin and 0.05% sodium azide [all from Sigma-Aldrich]) on ice, then washed twice with FACS buffer, and finally resuspended in FACS buffer. For competition assays, fucoidan from Fucus vesiculosus (Sigma; contained < 0.1 EU/µg by LAL assay) or mAbs against the 85-kDa subunit of CD91 (immunoglobulin G1 [IgG1], clone 5A6; Progen, Heidelberg, Germany), CD36 (clone SMO; Sigma-Aldrich), CD62L (clone TC1; Coulter Immunotech), or isotype controls (IgG1 or IgG2b; BD Pharmingen) were added 10 minutes prior to labeled Gp96. For intracellular stainings, the Cytofix/Cytoperm kit (BD Pharmingen) was used according to the manufacturer's instructions.All analyses were performed by FACScalibur and CellQuest (both from Becton Dickinson, Heidelberg, Germany) software. Phagocytosis For analysis of phagocytic activity, ingestion of PE-labeled polystyrene microspheres (diameter, 1 µm; Fluoresbrite Plain Microspheres PCRed, Polysciences, Warrington, PA) was evaluated as described elsewhere.25 Briefly, aliquots of 2 × 105 freshly purified PMNs were incubated in the presence of stimuli as indicated and 5 × 106 microbeads (E/T ratio, 1:25) for 45 to 120 minutes at 37°C, then kept on ice, washed twice in FACS buffer, and fixed in 1% paraformaldehyde in PBS. Analysis was performed by FACS.Detection of IL-8 production For analysis by enzyme-linked immunosorbent assay (ELISA), supernatants were derived from stimulation with either purified PMNs, PMNs with 5% MNCs added, or MNCs after 6 hours, and frozen at 20°C
until required. Supernatants were analyzed by ELISA for IL-8 (OptEIA;
BD Pharmingen) according to the manufacturer's instructions.
For intracellular cytokine stainings, PMNs or MNCs were incubated with indicated stimuli in the presence of brefeldin A (Sigma) or monensin (GolgiStop, BD Pharmingen), respectively, and subsequently labeled with FITC-conjugated anti-CD66b or CD14 and PE-labeled anti-IL-8 as described in "Antibodies and stainings for flow cytometry."
Interaction of Gp96-FITC with PMNs and monocytes We first investigated whether PMNs and monocytes are able to bind Gp96 specifically. Freshly isolated PBLs were incubated with FITC-labeled Gp96 with or without excess of unlabeled Gp96 or an irrelevant protein. FACS analysis showed that both PMNs and monocytes are able to bind FITC-labeled Gp96 (Figure 1). The specificity of Gp96-FITC binding was indicated by competition of binding in the presence of excess of unlabeled Gp96 (Figure 1A or C), but not of an irrelevant protein (Figure 1B or D). In addition, we found that the presence of unlabeled Gp96 or LPS as well as fucoidan, a known ligand for selectins or class A scavenger receptors,26,27 interfered with LPS-FITC binding on PMNs (Figure 1E). Binding of Gp96-FITC could be also competed by unlabeled LPS or fucoidan, but not by antibodies against CD36 or CD91 (data not shown), suggesting that other receptors are involved.
PMNs pretreated with phorbolester showed an increased binding capacity
for Gp96-FITC or LPS-FITC compared with unstimulated PMNs (Figure
2), indicating that the receptors for
both, Gp96 or LPS, are up-regulated (n = 5; for Gp96-FITC at 50 µg/mL, P < .05; for LPS-FITC at 30 µg/mL,
P < .001, by Student t test). Stimulating PMNs with phorbolester resulted in the enhanced
surface expression of CD66b and CD11b concurrent with a complete loss of L-selectin (CD62L). CD14 expression on PMNs was not altered (data
not shown). This suggests that neither L-selectin nor CD14 are the main
receptors for Gp96 or LPS on PMNs.
Gp96 increases phagocytic activity of PMNs The innate defense mechanisms of PMNs depend on their ability to ingest and subsequently eliminate pathogens by oxidative and nonoxidative processes. Antibody and complement receptors, as well as bacterial products such as LPS, mediate activation of human PMNs.22 To assess the influence of Gp96 on phagocytic activity of PMNs, we used a model of unopsonized fluorochrome-labeled polystyrene beads as targets, and phagocytosis was quantitated by FACS analysis.25 PMNs showed a low spontaneous phagocytosis of polystyrene beads in the absence of exogenous stimuli, but in the presence of Gp96 as well as LPS or phorbolester there was a marked enhancement of phagocytic activity (Figure 3). Even at low concentrations of Gp96 (10 µg/mL), there was an increase of phagocytosis detectable (n = 4; P < .05 by Student t test), suggesting that Gp96 is able to activate PMNs.
Production of IL-8 by PMNs and monocytes IL-8 is an important PMN-attracting chemokine released by a variety of cells, including PMNs and monocytes upon activation.21,22 In order to assess the production of IL-8 in PMNs and monocytes, supernatants of purified PMNs or mononuclear cells were analyzed by ELISA. We observed that high amounts of IL-8 are present after incubating these cells with Gp96 or LPS as control (Figure 4). To confirm that the cells were activated by Gp96 and not by potential endotoxin contaminations, flanking fractions of the same Gp96 purification were analyzed in parallel. No significant increase in IL-8 release was detected. To exclude an inhibitory effect of the flanking fractions themselves due to differences in the salt concentration, the fractions were spiked with LPS (10 ng/mL). PMNs and monocytes were activated to levels comparable with LPS in normal medium. These controls demonstrate that Gp96, and not potential endotoxin contaminations, is responsible for the observed release of IL-8.
Because the preparation of PMNs usually renders a purity of
approximately 95% to 98%, it could not be excluded that the
production of IL-8 was exclusively due to the presence of monocytes.
Therefore, we added 5% MNCs to the PMN preparation, resulting in an
increase of monocytes (CD14+ cells) from 0.9% to 1.2% by
FACS analysis (for comparison, the MNC preparation contained 23%
CD14+ cells; not shown). Increasing the number of monocytes
resulted in a 2- to 3-fold increase of the released amount of IL-8. To demonstrate that both PMNs and monocytes contribute to the release of
IL-8, we investigated the production of IL-8 by intracellular staining and FACS anaylsis. Here we found a remarkable increase of
intracellular IL-8 in monocytes after stimulation with Gp96 (Figure
5, right panel), but not in T or B cells
using CD3 and CD19 as markers, respectively (not shown). PMNs also
showed a slight increase in IL-8 (Figure 5, left panel), but to a
lesser extent. These experiments demonstrate that monocytes as well as PMNs contribute to the production of IL-8 after stimulation with Gp96.
The HSP Gp96 is normally localized in the ER of cells and released into the extracellular space during necrotic cell death,5 (ie, after cell damage due to viral infection6 or excessive trauma [S. Herter et al, manuscript in preparation]). Extracellular Gp96 activates dendritic cells and macrophages by releasing inflammatory cytokines and inducing DC maturation.5,13 This feature, combined with the ability to transfer antigenic peptides for their MHC class I-restricted presentation,2,3 allows Gp96, together with other HSPs such as HSP70 and HSP90, to function as an efficient messaging system alerting the organism to bacterial or viral infection and possibly injury. Given the fact that during the early phase of inflammation, in particular, the majority of cells recruited to an inflammatory focus are mainly PMNs and monocytes,20 it can be assumed that these cells will encounter contact with Gp96 early on in inflammation. Therefore, we investigated whether PMNs and monocytes, in addition to macrophages and DCs, are able to interact with Gp96. We found that both cell types bind FITC-labeled Gp96. The binding of labeled Gp96 could be competed with excess of unlabeled Gp96, but not in the presence of the same amount of an irrelevant protein (Figure 1), suggesting that Gp96 is able to specifically interact with monocytes as well as PMNs. The In addition to the binding of Gp96 to PMNs and monocytes, we also examined whether Gp96 can mediate activation of these cells. PMNs play a crucial part in innate host defense with their ability to accumulate rapidly and to clear an inflammatory site by using their potent oxidative and nonoxidative effector mechanisms. One important function is the ingestion of particles (ie, bacteria and fungi) by phagocytosis.20 Therefore, we assessed the phagocytic activity of PMNs and found that the presence of Gp96 greatly enhances the phagocytic activity of these cells, even at low concentrations of Gp96 (Figure 3). Thus, Gp96 may represent an additional signal for PMNs to clear an inflammatory site. IL-8 is one of the first chemokines to be described; it was originally identified as a strong chemoattracting agent for PMNs produced by LPS-stimulated monocytes.32 In addition to its chemotactic effects, IL-8 also mediates activation for PMNs directly by enhancing degranulation.22 There is broad evidence to demonstrate the importance of IL-8 in leukocyte trafficking homeostasis and also in disease.33 We were able to show that both PMNs and monocytes produce IL-8 upon activation by Gp96 (Figure 4). Despite similar binding capacity for Gp96 of both cell types (Figure 1) and the ability to produce IL-8 after stimulation with Gp96 (Figure 5), there is a marked difference in the amount of IL-8 produced by PMNs or monocytes (Figure 4). We propose that Gp96 induces monocytes to attract PMNs to an inflammatory site via IL-8 secretion and that PMNs clear the site by enhanced phagocytic activity. Whether the phagocytic activity is mediated by Gp96 directly via receptor-ligand interactions on PMNs or indirectly by activation via IL-8 is not yet clear and will be the subject of further studies. In conclusion, Gp96 binds specifically to both PMNs and monocytes and mediates the release of large amounts of IL-8 by both cell types. Furthermore, we show that in addition to the production of IL-8, Gp96 also stimulates the phagocytic activity of PMNs, possibly to clear a site of cellular debris or microbial particles. Altogether, this work supports the notion that Gp96 acts as a danger signal to the innate immune system, now including also monocytes and PMNs as cell types responsive to Gp96. However, there are probably differences in the receptors involved in Gp96-mediated activation of monocytes and PMNs, the latter expressing neither CD36 nor CD91. Further studies will be necessary to elucidate the receptors and signaling pathways for Gp96-mediated interactions.
We thank Dr Birgitt Schönfisch from the Department of Biomathematics, University of Tübingen (Germany) for the support concerning the statistical analyses of the data.
Submitted July 25, 2002; accepted November 13, 2002.
Prepublished online as Blood First Edition Paper, November 21, 2002; DOI 10.1182/blood-2002-07-2261.
Supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 510, C1 to H.S.), the European Union (EC Project QLK3-CT-1999-00064), and the University of Tübingen (AKF- and IZKF-Programm).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hansjoerg Schild, Institute for Cell Biology, Department Immunology, Auf der Morgenstelle 15, D-72076 Tübingen, Germany; e-mail: hansjoerg.schild{at}uni-tuebingen.de.
1. Melnick J, Dul JL, Argon Y. Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum. Nature. 1994;370:373-375[CrossRef][Medline] [Order article via Infotrieve]. 2. Lammert E, Arnold D, Nijenhuis M, et al. The endoplasmic reticulum-resident stress protein gp96 binds peptides translocated by TAP. Eur J Immunol. 1997;2:923-927.
3.
Singh-Jasuja H, Toes RE, Spee P, et al.
Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis.
J Exp Med.
2000;191:1965-1974 4. Spee P, Neefjes J. TAP-translocated peptides specifically bind proteins in the endoplasmic reticulum, including gp96, protein disulfide isomerase and calreticulin. Eur J Immunol. 1997;27:2441-2449[Medline] [Order article via Infotrieve].
5.
Basu S, Binder RJ, Suto R, Anderson KM, Srivastava PK.
Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway.
Int Immunol.
2000;12:1539-1546
6.
Berwin B, Reed RC, Nicchitta CV.
Virally induced lytic cell death elicits the release of immunogenic GRP94/gp96.
J Biol Chem.
2001;276:21083-21088 7. Udono H, Srivastava PK. Comparison of tumor-specific immunogenicities of stress-induced proteins gp96, hsp90, and hsp70. J Immunol. 1994;152:5398-5403[Abstract].
8.
Tamura Y, Peng P, Liu K, Daou M, Srivastava PK.
Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations.
Science.
1997;278:117-120
9.
Arnold-Schild D, Hanau D, Spehner D, et al.
Cutting edge: receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells.
J Immunol.
1999;162:3757-3760
10.
Bevan MJ.
Minor H antigens introduced on H-2 different stimulating cells cross-react at the cytotoxic T cell level during in vivo priming.
J Immunol.
1976;117:2233-2238 11. Carbone FR, Kurts C, Bennett SR, Miller JF, Heath WR. Cross-presentation: a general mechanism for CTL immunity and tolerance. Immunol Today. 1998;19:368-373[CrossRef][Medline] [Order article via Infotrieve]. 12. Sigal LJ, Crotty S, Andino R, Rock KL. Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen. Nature. 1999;398:77-80[CrossRef][Medline] [Order article via Infotrieve]. 13. Singh-Jasuja H, Scherer HU, Hilf N, et al. The heat shock protein gp96 induces maturation of dendritic cells and down-regulation of its receptor. Eur J Immunol. 2000;30:2211-2215[Medline] [Order article via Infotrieve]. 14. Binder RJ, Han DK, Srivastava PK. CD91: a receptor for heat shock protein gp96. Nat Immunol. 2000;1:151-155[CrossRef][Medline] [Order article via Infotrieve].
15.
Berwin B, Hart JP, Pizzo SV, Nicchitta CV.
Cutting edge: CD91-independent cross-presentation of GRP94 (gp96)-associated peptides.
J Immunol.
2002;168:4282-4286 16. Panjwani N, Popova L, Febbraio M, Srivastava PK. The CD36 scavenger receptor as a receptor for Gp96 [abstract]. Cell Stress Chaperones. 2000;5:391. 17. Randow F, Seed B. Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability. Nat Cell Biol. 2001;3:891-896[CrossRef][Medline] [Order article via Infotrieve].
18.
Vabulas RM, Braedel S, Hilf N, et al.
The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway.
J Biol Chem.
2002;277:20847-20853
19.
Hilf N, Singh-Jasuja H, Schwarzmaier P, Gouttefangeas C, Rammensee HG, Schild H.
Human platelets express heat shock protein receptors and regulate dendritic cell maturation.
Blood.
2002;99:3676-3682 20. Page AR, Good RA. A clinical and experimental study of the function of neutrophils in the inflammatory response. Am J Pathol. 1958;34:645-669[Medline] [Order article via Infotrieve]. 21. Lloyd AR, Oppenheim JJ. Poly's lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response. Immunol Today. 1992;13:169-172[CrossRef][Medline] [Order article via Infotrieve].
22.
Ben-Baruch A, Michiel DF, Oppenheim JJ.
Signals and receptors involved in recruitment of inflammatory cells.
J Biol Chem.
1995;270:11703-11706 23. Van Furth R, Diesselhoff-den Dulk MC, Mattie H. Quantitative study on the production and kinetics of mononuclear phagocytes during an acute inflammatory reaction. J Exp Med. 1973;138:1314-1330[Abstract]. 24. Radsak M, Iking-Konert C, Stegmaier S, Andrassy K, Hänsch GM. Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation. Immunology. 2000;101:521-530[CrossRef][Medline] [Order article via Infotrieve]. 25. Lehmann AK, Sornes S, Halstensen A. Phagocytosis: measurement by flow cytometry. J Immunol Methods. 2000;243:229-242[CrossRef][Medline] [Order article via Infotrieve]. 26. Malhotra R, Priest R, Bird MI. Role for L-selectin in lipopolysaccheride-induced activation of neutrophils. Biochem J. 1996;320:589-593.
27.
Maxeiner H, Husemann J, Thomas CA, Loike JD, El Khoury J, Silverstein SC.
Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized low-density lipoproteins and in secretion of H2O2.
J Exp Med.
1998;188:2257-2265
28.
Armesilla AL, Vega MA.
Structural organization of the gene for human CD36 glycoprotein.
J Biol Chem.
1994;269:18985-18991
29.
Kurt-Jones EA, Mandell L, Whitney C, et al.
Role of Toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils.
Blood.
2002;100:1860-1868
30.
Haziot A, Hijiya N, Gangloff SC, Silver J, Goyert SM.
Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccheride in CD14-deficient and Toll-like receptor 4-deficient mice.
J Immunol.
2001;166:1075-1078
31.
Li M, Carpio DF, Zheng Y, et al.
An essential role of the NF-kappaB/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells.
J Immunol.
2001;166:7128-7135
32.
Peveri P, Walz A, Dewald B, Baggiolini M.
A novel neutrophil-activating factor produced by human mononuclear phagocytes.
J Exp Med.
1988;167:1547-1559 33. Baggiolini M. Chemokines in pathology and medicine. J Intern Med. 2001;250:91-104[CrossRef][Medline] [Order article via Infotrieve].
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  T. Warger, N. Hilf, G. Rechtsteiner, P. Haselmayer, D. M. Carrick, H. Jonuleit, P. von Landenberg, H.-G. Rammensee, C. V. Nicchitta, M. P. Radsak, et al. Interaction of TLR2 and TLR4 Ligands with the N-terminal Domain of Gp96 Amplifies Innate and Adaptive Immune Responses J. Biol. Chem., August 11, 2006; 281(32): 22545 - 22553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Xia, F. Hou, J. Li, Y. Ke, and H. Nie Two novel proteins bind specifically to trichosanthin on choriocarcinoma cell membrane. J. Biochem., April 1, 2006; 139(4): 725 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Wang, J. T. Kovalchin, P. Muhlenkamp, and R. Y. Chandawarkar Exogenous heat shock protein 70 binds macrophage lipid raft microdomain and stimulates phagocytosis, processing, and MHC-II presentation of antigens Blood, February 15, 2006; 107(4): 1636 - 1642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Cassatella, V. Huber, F. Calzetti, D. Margotto, N. Tamassia, G. Peri, A. Mantovani, L. Rivoltini, and C. Tecchio Interferon-activated neutrophils store a TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable following exposure to proinflammatory mediators J. Leukoc. Biol., January 1, 2006; 79(1): 123 - 132. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Massa, C. Melani, and M. P. Colombo Chaperon and Adjuvant Activity of hsp70: Different Natural Killer Requirement for Cross-Priming of Chaperoned and Bystander Antigens Cancer Res., September 1, 2005; 65(17): 7942 - 7949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Pilla, P. Squarcina, J. Coppa, V. Mazzaferro, V. Huber, D. Pende, C. Maccalli, G. Sovena, L. Mariani, C. Castelli, et al. Natural Killer and NK-Like T-Cell Activation in Colorectal Carcinoma Patients Treated with Autologous Tumor-Derived Heat Shock Protein 96 Cancer Res., May 1, 2005; 65(9): 3942 - 3949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Berwin, Y. Delneste, R. V. Lovingood, S. R. Post, and S. V. Pizzo SREC-I, a Type F Scavenger Receptor, Is an Endocytic Receptor for Calreticulin J. Biol. Chem., December 3, 2004; 279(49): 51250 - 51257. [Abstract] [Full Text] [P |
Top
Abstract
Introduction
(TNF-
) or PMNs prestimulated with PMA (10 ng/mL) for
10 minutes at 37°C (
) were incubated with the indicated
concentrations of gp96-FITC (A) or LPS-FITC (B) and analyzed by FACS.
The increase in mean fluorescence was statistically significant for
both Gp96-FITC (50 µg/mL) or LPS-FITC (30 µg/mL) binding in 5 independent experiments (n = 5; P < .05 or
P < .001, respectively, by Student t
test).
), PMNs with 5% MNCs added (
) (A) or
MNCs only (B) were analyzed. All samples were taken in duplicates. The
results shown are representative of 3 independent experiments.
Statistical analysis of all 3 experiments indicated a significant
increase in the release of IL-8 in the presence of Gp96 (at 100 µg/mL
or 20 µg/mL for PMNs, P < .001; for PMNs+MNC or MNCs
alone, P < .05) or LPS (at 10 ng/mL for PMNs,
P = .0017; for PMNs+MNCs or MNCs, P < .05;
flanking fraction+LPS, 10 ng/mL for PMNs, P < .001; for
MNCs, P < .05), but not for flanking fraction alone (by
Student t test).
