From the Department of Biology, University of Patras, Patras 265 00, Greece
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ABSTRACT |
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Insect hemocytes in response to
lipopolysaccharide (LPS) of Gram-negative bacteria facilitate binding
and internalization of either cell-associated or cell-free LPS
(Charalambidis, N. D., Foukas L. C., and Marmaras V. J. (1996) Eur. J. Biochem. 236, 200-206). An early event
in LPS signaling in hemocytes involves protein tyrosine phosphorylation
(Charalambidis N. D., Zervas C. G., Lambropoulou M., Katsoris
P. G., and Marmaras V. J.(1995) Eur. J. Cell
Biol. 67, 32-41). Here we report further data of LPS-mediated
signal transduction responsible for Escherichia coli phagocytosis. We demonstrate that both adhesion of hemocytes to substrata and LPS stimulation can cause activation of
p44MAPK in Ceratitis capitata hemocytes but
with distinct kinetics indicating different functions. In addition, we
showed that Drk, a homolog protein to the mammalian GRB2, is implicated
in the transmission of LPS signaling, indicating that the
Ras/mitogen-activated protein kinase pathway is involved. Either the
cell-free or the cell-associated LPS appears to attach to the hemocyte
surface by the same mechanism that is based on the cross-linking of LPS
to membrane-associated p47 via the intermediacy of tyrosine derivatives
generated by the action of phenol oxidase. By contrast, the cell-free
LPS internalization into the hemocytes differs from the cell-associated
LPS internalization. For E. coli internalization integrin
receptors as well as cytoskeletal rearrangements are required, as
judged by inhibition of E. coli internalization in the
presence of the RGD peptide, 3-integrin antibodies, and
cytochalasin D.
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INTRODUCTION |
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Phagocytosis is a process that involves binding and internalization of pathogens, and it is essential for host defense in higher eukaryotes. Phagocytosis in mammals is mainly achieved by neutrophils, monocytes, and macrophages, whereas in insects it is achieved by granular cells and plasmatocytes (1, 2). Even though phagocytosis is an event of major importance for insect immunity, the biochemical pathways involved in it are poorly understood.
Recently, substantial progress in understanding the mechanism of LPS1 binding and Escherichia coli entrapment as well as nodule formation has been achieved by Ceratitis capitata hemocytes. Upon LPS triggering, a protein, p47, is released from hemocytes, in vitro, which together with tyrosine and phenol oxidase cause aggregation of E. coli cells (3, 4). The aggregation is based on the cross-linking of LPS to p47 via the intermediacy of tyrosine derivatives generated by the action of phenol oxidase (5). This protein is also weakly associated with the hemocyte surface (4) and covalently binds to LPS with the same mechanism. The LPS·catechol·p47 complex is then internalized into hemocytes, as judged by proteinase K treatment (5). The key regulatory component for the complex formation is pro (phenol oxidase)-activators (serine proteases), which are released by hemocytes upon LPS triggering and activate the hemocyte surface phenol oxidase (6). The process of LPS-stimulated release of p47 and pro-activators (phenol oxidase) depends on protein tyrosine phosphorylation (4, 6).
Mammalian macrophages, on the other hand, utilize either the
LPS-binding protein and membrane CD14 for LPS binding and
internalization or the alternative complement C3 pathway. LPS forms a
ternary complex, LPS binding protein·LPS· membrane CD14 based
on weak interactions. This complex is utilized for LPS internalization (7). LPS-stimulated macrophages cause the activation of MAP kinases and
the release of active molecules, such as tumor necrosis factor- and
interleukin-1
(8, 9). However, it is not known whether the signal is
transduced through G proteins or through the Ras protein (10). In
insects, MAP kinases have only been detected in Drosophila
and are implicated in the development of the compound eye (11).
Recently, the Drosophila MAP kinase DJNK, which is
homologous to the mammalian c-Jun amino-terminal kinase, has been
reported to participate in multiple physiological processes. The DJNK
signaling pathway mediates morphogenesis and initiates an insect immune
response upon LPS triggering (12).
Internalization of a pathogen by a phagocyte requires multiple successive interactions between the phagocyte and the target (1). Internalization is largely achieved by members of the integrin family, a class of heterodimeric surface receptors expressed on almost all cells in higher eukaryotes. Integrins act not only as simple mediators of cell adhesion but can also transduce signals across the cell membranes (13). Integrin-mediated signals activate cellular proteins, including most prominently MAP and FAK kinases (14). In vertebrates, integrins are important for processes such as embryonic morphogenesis, leukocyte migration, platelet aggregation, and regulation of cell proliferation and differentiation (1, 14). Concerning insects, integrins have so far only been characterized in Drosophila melanogaster and are known to be required for a host of morphogenetic units (2).
The above results prompted us to clarify further the signal
transduction pathways leading to E. coli phagocytosis by
hemocytes in response to LPS. In the present work we show that the
Ras/MAP kinase signal transduction pathway plays a pivotal role in the immune responses of insect hemocytes upon LPS triggering. In addition, cell-free LPS is evidently internalized into hemocytes by a
receptor-mediated endocytosis, whereas E. coli is
phagocytosed via a 3 integrin-dependent process.
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EXPERIMENTAL PROCEDURES |
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Materials--
LPS (from E. coli, serotype 0111:B4)
was purchased from Sigma. Anti-p47 and anti-phenol oxidase are rabbit
polyclonal antisera specific for C. capitata p47 and phenol
oxidase, respectively, raised at our laboratory, as described
previously (5, 15). Rabbit polyclonal antiserum against the
Drosophila Drk protein, was kindly provided by Dr. T. Pawson
(University of Toronto). ERK1 (K-23), an affinity purified rabbit
polyclonal antibody raised against a peptide that corresponds to amino
acids 305-327 within the XI subdomain of the rat ERK1 MAP kinase, was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
anti-phosphotyrosine monoclonal antibody PY20 and the
anti-3 monoclonal antibody raised against a peptide
corresponding to amino acids 16-223 of the extracellular domain of the
mouse
3 integrin subunit, as well as the A431 and human
fibroblasts cell lysates, were purchased from Transduction Laboratories
(Lexington, KY). Anti-human HLA DR monoclonal antibody was from
Boehringer Mannheim (Mannheim, Germany). 96-Well cell culture plates
were from Corning Glass Works (Corning, NY), and 8-well glass slides
were from Nunc, Inc. (Naperville, IL). Other materials were obtained as
indicated and were of the highest grade available.
Isolation, Culture, and Homogenization of C. capitata Larval Tissues-- C. capitata were reared as described previously (16). Isolation and homogenization of larval hemocytes, fat bodies, and integuments were performed as described elsewhere (6). Protein was assayed by the method of Bradford (17).
Hemocyte monolayers were prepared by allowing 105 cells to adhere on cell culture wells for 15 min at 25 °C, followed by washing with 100 µl of insect Ringer's solution (128 mM NaCl, 18 mM CaCl2, 1.3 mM KCl, 2.3 mM NaHCO3, pH 7), for 3 times to remove nonadherent hemocytes. Hemocyte monolayers were cultured in Grace's insect medium (Sigma).Cell Attachment-mediated and LPS-induced Tyrosine Phosphorylation of Hemocyte Proteins-- Isolated hemocytes were suspended in Grace's insect medium and then either incubated nonadherently in a rotator or plated onto cell culture wells (105 cells per well) at 25 °C for the indicated times. For LPS stimulation, hemocyte monolayers, which had been cultured for 2 h at 25 °C, were stimulated with 100 µg/ml LPS for 5 min at 25 °C. In both cases, after incubations, hemocytes were washed twice with cold Ringer's solution, then lysed in boiling electrophoresis sample buffer (60 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (v/v) SDS, 10 mM dithiothreitol), and processed for immunoblot analysis.
Osmotic Loading of Drk Antibodies and Evaluation of Its Effect in LPS-stimulated p47 Exocytosis-- Intracellular loading of Drk antibodies or control antibodies or bovine serum albumin was achieved by promoting uptake of extracellular proteins by incubation in a hypertonic medium, followed by lysis of cytoplasmic pinosomes in a hypotonic solution by the method of Okada and Rechsteiner (18). Briefly, hemocyte monolayers, prepared as described above, were washed with Ringer's solution and then incubated for 10 min in a hypertonic medium containing 0.5 M sucrose, 10% (w/v) polyethylene glycol 1,000, and Drk IgGs, DR antibody, or bovine serum albumin (30 µg/ml) in Grace's insect medium. The monolayers were then rinsed with a hypotonic solution of diluted Grace's medium/water (6:4) and incubated in the hypotonic medium for 2 min. After rinsing 3 times with normal Grace's medium, the monolayers were allowed to recover for 2 h before further procedures were performed. Viability of the hemocytes was assessed by exclusion of trypan blue dye (Sigma).
After loading and cell recovery, hemocytes were stimulated with 100 µg/ml LPS for 5 min and then washed and allowed to secrete for 1 h. After that, culture media were collected, analyzed in SDS-PAGE, and immunoblotted against p47.SDS-PAGE and Immunoblot Analysis--
SDS-PAGE was performed
according to Laemmli (19) in 12% acrylamide, 0.12% bisacrylamide slab
gels (17). After gel separation, the proteins were transferred
electrophoretically onto Immobilon P polyvinylidene fluoride membranes
(Millipore Corp., Bedford, MA) according to Towbin et al.
(20). Membranes were then incubated in TBS (10 mM Tris-HCl,
pH 7.5, 100 mM NaCl) containing 5% (w/v) bovine serum
albumin and 0.05% (v/v) Tween 20 for 1 h at room temperature.
Subsequently, the membranes were incubated with primary antibody in the
appropriate concentration (0.5 µg/ml for anti-rat ERK1,
anti-3 integrin, and PY20 antibodies or 1:2,000 and
1:10,000 dilution of anti-Drk and anti-p47 antisera, respectively) in
TBS containing 1% (w/v) bovine serum albumin and 0.05% (v/v) Tween 20 for 1 h at room temperature. Membranes were washed 3 times for 10 min in TBS containing 0.05% (v/v) Tween 20, followed by incubation
with goat anti-mouse or anti-rabbit IgG peroxidase conjugates
(Transduction Laboratories) diluted 1:4,000. Immunoreactivity was
detected by incubating the membranes in TBS containing 0.06% (w/v)
diaminobenzidine, 0.018% NiCl2 (w/v), 0.3%
H2O2 (v/v). In the case of anti-phosphotyrosine
immunoblotting, antigen-antibody complexes were visualized on x-ray
film by enhanced chemiluminescence (ECL) methodology (Amersham
Pharmacia Biotech). Stripping of membranes after ECL detection was
performed according to the manufacturer (Amersham Pharmacia
Biotech).
Assays for LPS Binding and Internalization into the
Hemocytes--
Formation of the radiolabeled LPS-catechol adduct
(LPS-Y) and the assays of LPS-Y binding and internalization into the
hemocytes were performed as described previously (5). For inhibition experiments, hemocyte monolayers were first incubated in 100 µl/well of 1 µg/ml anti-3, or 2 mM RGDS peptide
(Sigma), or 10 µM cytochalasin D (Sigma) diluted in
Grace's insect medium for 30 min at 25 °C before the addition of
the labeled LPS-Y complex.
Phagocytosis Assay-- 200-µl aliquots of hemocytes suspended in Grace's medium (0.5 × 106 cells/ml) were added into the wells of a multiwell glass LabTeK chamber (Nunc). After allowing 15 min for settlement and attachment of hemocytes, monolayers were washed three times in Grace's medium to remove nonadherent cells and challenged with E. coli or latex particles (0.8 µm diameter, Sigma). The phagocytic activity of E. coli was determined by a fluorescence quenching assay with trypan blue. The assay is a modification of a method originally established for vertebrate blood cells to distinguish ingested from attached bacteria (21). In brief, 50 µl of fluorescein isothiocyanate-labeled E. coli suspension (~1 × 108 E. coli) was added into 200 µl of monolayers (ratio E. coli/hemocyte, 20:1). After incubation for 2 h at 31 °C the non-adhering E. coli were removed by exchanging the supernatants three times by Grace's medium. 250 µl of the supernatant in each well was exchanged by a trypan blue solution (2 mg/ml in Grace's medium). 20 min later the cells were washed with culture medium and fixed with a 4% formaldehyde solution for 10 min. Trypan blue quenches only non-phagocytosed bacteria. Phagocytosis was observed by fluorescent microscopy. The phagocytic activity of latex partially was determined by the phase contrast microscope; under the microscope the particles appear refringent.
For inhibition experiments hemocyte monolayers were preincubated in 100 µl/cell of genistein or anti-phenol oxidase or anti- ![]() |
RESULTS |
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Hemocytes Possess Putative Tyrosine-phosphorylated Proteins-- Insect hemocytes require protein tyrosine phosphorylation in order to attach and internalize cell-free or cell-associated LPS (3-5).
To elucidate the triggered LPS signaling pathway which leads to phagocytosis of bacteria, we identified the tyrosine-phosphorylated polypeptides present in hemocyte extracts. Immunoblot analysis using the monoclonal antibody PY20 and ECL as detection method showed a number of phosphorylated proteins (Fig. 1). Control experiments where antibodies were preincubated either with tyrosine phosphate or with a mixture of serine/threonine phosphates demonstrated the specificity of tyrosine-phosphorylated proteins.
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Cell Adhesion and LPS-stimulated Activation of p44MAPK-- It is well known that hemocytes normally adhere to and spread upon foreign surfaces in protein-free medium (22). In our experiments, the adherence of hemocytes in the absence of any stimulus was relatively constant under the conditions of the assay, with an average of 70% in 15 min. Furthermore, it is well known that cell adhesion affects the pattern of tyrosine-phosphorylated proteins in cells (23, 24). To study the putative pattern of tyrosine-phosphorylated proteins upon LPS triggering, three series of experiments were performed. In the first one series, hemocytes were kept in suspension (as control for plated cells); in the second series, hemocytes were plated in the absence of any stimulator (as control for LPS-triggered); and in the third series, monolayers were treated with LPS and incubated for the times indicated in the figure legends.
As shown in Fig. 2, only a few proteins were significantly phosphorylated on tyrosine, including a protein with molecular mass of 44 kDa (identified as MAP kinase, see below). Although some variation was observed between different experiments due to a rather low level of activation of hemocytes inherent in the preparation of isolated hemocytes, the most important difference, constant in all experiments, was that observed between hemocytes in suspension and attached cells. Cells in suspension showed a progressive dephosphorylation in relation to incubation time, presumably due to activation of hemocytes during the isolation procedure. By contrast, attached hemocytes showed a progressive increase in protein phosphorylation due to the adherence effect.
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Drk Plays an Essential Role in Immune Responses-- As MAP kinases are considered to be key molecules for the convergence of extracellular signals, we sought to elucidate an upstream component in the pathway leading to the MAP kinase activation. A key component that functions downstream of the tyrosine kinases and upstream of the MAP kinase pathway is Drk, which is homologous to the mammalian GRB2 (27).
We initially tried to identify the presence of the Drk protein in C. capitata larval tissues. Immunoblot analysis by using polyclonal anti-Drk antibodies (Fig. 5) clearly showed a 24-kDa polypeptide recognized by anti-Drk in the tissues tested. Furthermore, to determine whether this SH2-SH3 adaptor protein is implicated in the transmission of LPS signaling, we used osmotic lysis of pinocytic vesicles to load hemocytes with anti-Drk. Fig. 6A showed the successive uptake of anti-Drk IgGs by the hemocytes. Furthermore, to determine whether the LPS signaling is directly affected by Drk, we examined whether the p47 immune protein was released or not after LPS stimulation of the antibody-loaded hemocytes. Fig. 6B showed that in hemocytes loaded with anti-Drk, the LPS-induced release of the immune protein p47 was largely inhibited (Fig. 6, lanes 9 and 10) compared with the unloaded hemocytes (Fig. 6, lanes 3 and 4), which reached the level of basal secretion (Fig. 6, lanes 1 and 2). The specificity of the anti-Drk effect was checked by loading the hemocytes with bovine serum albumin (Fig. 6, lanes 7 and 8) or with the monoclonal anti-DR, an irrelevant antibody (Fig. 6, lanes 5 and 6). Consequently, in hemocytes the Ras/MAP kinase pathway is activated in response to endotoxic LPS.
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Phagocytosis and 3 Integrin--
Phagocytosis is a
multistep process including, among others, attachment and
internalization of invaders. In the following experiments, an attempt
was undertaken to record some of the requirements needed for the
process of E. coli phagocytosis by hemocytes and to compare
it with the known requirements for cell-free LPS internalization into
hemocytes (4-6). The phagocytic activity of the hemocytes against both
latex beads and heat-fixed E. coli cells was evaluated by a
fluorescence quenching assay with trypan blue (21). In both cases about
13% of the cells kept at 31 °C were phagocytically active.
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DISCUSSION |
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Phagocytosis of bacteria usually elicits a more complex and thus different response than LPS alone. Therefore, it was interesting to examine whether the biochemistry of cell-free and cell-associated LPS attachment and internalization differ.
Our current findings demonstrated that initially, both cell-free LPS and E. coli cells recognize p47 on the hemocyte surface (5). This process is associated with phosphorylation of intracellular proteins (4, 5), representing a potential signal recognition pathway leading to the release of intracellular p47 and pro-activators (phenol oxidase) from hemocytes (4, 6), and presumably also the release of certain effector molecules responsible for both cell adhesion and cell spreading. The release upon LPS triggering p47 and pro-activators (phenol oxidase) is responsible for the association of both cell-free and cell-associated LPS (E. coli) with hemocyte surface (4-6). The process of attachment, therefore, involves a signal transduction pathway (4, 5). In mammals, at present, there is no evidence that signaling is required for LPS attachment and/or internalization nor there is evidence that it is not dependent upon some sort of signal to the cell (8, 9).
The fact that intracellular signaling pathways are required for LPS attachment to the hemocyte surface prompted us to investigate further, in order to clarify the intracellular LPS-induced signal transduction pathways. We have demonstrated that LPS can cause a strong and prompt activation of p44MAPK in hemocytes (Fig. 4). This activation of p44MAPK differs from that caused by adhesion of hemocytes to substrata (Fig. 2), as the kinetics of adhesion-mediated activation of p44MAPK and LPS-mediated activation are distinct indicating different functions. Therefore, it appears that LPS-mediated activation of p44MAPK is independent of cell adhesion-mediated activation of p44MAPK, which as it is well known is based on integrins (24, 25). Enhancement of p44MAPK activity has also been demonstrated in mammalian macrophages upon LPS triggering (10).
The notion that the Ras/MAP kinases pathway is considered to be a key pathway for the convergence of extracellular signals, and their transmission throughout the cell, encouraged us to search for an upstream component in the pathway leading to MAP kinase activation, in order to establish the hypothesis that this pathway is involved in the outside-in signaling by LPS. Indeed, we succeeded in demonstrating that Drk, a downstream receptor kinases molecule, is present in C. capitata hemocytes (Fig. 3). In addition, hemocytes loaded with anti-Drk inhibited the release of immune proteins (3, 5), suggesting the involvement of Drk in LPS signaling. This molecule has so far only been characterized in Drosophila and is homologous to GRB2 of mammalian cells (27).
These results strongly support that p44MAPK and the effector molecule Drk are evidently involved in insect hemocyte immune responses. Therefore, it can be concluded that LPS signaling for the release of immune proteins is transmitted via the Ras/MAP kinases pathway. The Ras/MAP kinases pathway has not been implicated in any aspect of the invertebrate immunity nor in macrophage immune responses (2). Evidently, much more work is needed to elucidate in detail the signaling pathways leading to the release of immune proteins. Furthermore, it would be essential to see whether Ras/MAP kinases pathway is normally involved in the hemocyte immune responses challenged by other elicitors or other unrelated microbes.
The LPS signaling for the release of immune proteins transmitted via the Ras/MAP pathway is essential for the attachment of both cell-free or cell-associated LPS at the hemocyte surface. The mechanism of attachment for both LPS forms, as can be evaluated from the results of Table I, is the same. That is, both LPS forms cross-link to hemocyte surface p47 via the intermediacy of tyrosine derivatives generated by the action of phenol oxidase, as is the case for cuticular protein-chitin cross-links during sclerotization (28). This attachment is a prerequisite for both LPS forms internalization into hemocytes (5).
The sum of our data (Refs. 4-6 and the present results), however, strongly supports that cell-free LPS internalization into hemocytes differs from cell-associated LPS internalization. As criteria for the process of internalization were the resistance of cell-free LPS binding to dissociation by proteinase K and the phagocytic activity of the hemocytes against E. coli cells, as evaluated by an established fluorescence-quenching assay.
The fact that the phagocytic activity was largely inhibited, when
hemocytes were preincubated with RGD peptides or anti-3 (Table I) and that
3 integrins are expressed on hemocyte
surface (Fig. 7), raises the possibility that
3
integrins might be implicated in insect immunity. Integrins in insects
have so far also been identified in D. melanogaster (1, 29).
Considering what is known about integrins and their roles in
phagocytosis, as well as the results of Table I and Fig. 7, we can
propose that
3 integrin might be responsible for
E. coli internalization without affecting attachment.
Similar results have already been obtained in other systems. Integrins
v
3 and
v
5
promote adenovirus internalization without affecting attachment
(30).
Additional strong evidence supporting the notion that the biochemistry
of cell-free LPS internalization differs from E. coli internalization are the results with cytochalasin D (Table I). LPS-mediated activation of p44MAPK is not affected by
cytochalasin D treatment, indicating that these kinases are not
associated with cytoskeleton rearrangements and, therefore, are
responsible for E. coli attachment. In addition, cytochalasin D does not affect the internalization of cell-free LPS,
whereas phagocytosis of E. coli cells is completely
inhibited (Table I), indicating that cytoskeletal rearrangements are
essential for phagocytosis, and therefore, 3 integrin is
responsible for E. coli internalization.
In summary, cell-free and cell-associated LPS are recognized and attached to the hemocyte surface through the proposed mechanism by utilizing the Ras/MAP kinase pathway. However, the process of internalization differs. For E. coli internalization, integrin receptors, as well as cytoskeletal rearrangements, are also required. It remains to be elucidated how the E. coli cells bind and activate integrin-specific signals for their internalization.
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FOOTNOTES |
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* This work was supported by a grant from the Ministry of Development, General Secretariat for Research and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence and reprint requests should be addressed.
Tel.: 30-61-997607; Fax: 30-61-994797; E-mail: marmaras{at}patreas.patras.gr.
1 The abbreviations used are: LPS, lipopolysaccharide; MAP, mitogen-activated protein; ERK, extracellular signal-regulated protein kinase; Drk, downstream of receptor kinase; mAb, monoclonal antibody PAGE, polyacrylamide gel electrophoresis; Y, catechol; TBS, Tris-buffered saline;membrane CD14.
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REFERENCES |
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