| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Biomedical and Physiological Research Group, University of Wales Swansea, Singleton Park, Swansea, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In invertebrates, including insects, due to the lack of Ab-based immunity, lectins probably play a major role in nonself recognition, with several reports of endogenous serum lectins having opsonic activity for invading pathogens (e.g., 9–14). If, however, lectins act in an analogous manner to Abs, then it is essential that multiple lectins are present in the circulation, each with different carbohydrate-binding specificities, and thus able to recognize a wide variety of invading organisms. To date, multiple lectins have been purified from only three species of insects, namely, the silkworm, Bombyx mori (15, 16); the American cockroach, Periplaneta americana (17, 18, 19, 20, 21); and the West Indian leaf cockroach, Blaberus discoidalis (22, 23). Two of the lectins from P. americana, i.e., the LPS-binding protein (11) and the Periplaneta lectin (14), have been reported to bind to, and increase the in vivo clearance of Escherichia coli from the hemolymph. In B. discoidalis, at least four lectins occur, namely, BDL1, BDL2, BDL3, and GSL, of which three, BDL1, BDL2 (22), and GSL (23), have been purified. Of these lectins, BDL1 has been shown to have properties similar to MBLs in terms of specificity, structure, and activation of complement (our unpublished data), and GSL has been shown to be similar to invertebrate C-reactive proteins.5 These endogenous lectins are also capable of enhancing the activation of the prophenoloxidase system (24), a melanization cascade that is an important component of the insect immune defense system involved in numerous defense reactions, including encapsulation, nodulation, and phagocytosis (25).
The present study examines in detail, for the first time in an invertebrate, the role of multiple, endogenous lectins in immune recognition, in particular, the effects of the four endogenous serum lectins from B. discoidalis, and four exogenous lectins of comparable carbohydrate specificity, on the in vitro phagocytosis of representatives from three major groups of microorganisms, namely yeast and Gram-positive and Gram-negative bacteria. Each of these microorganisms has very different surface carbohydrate properties (26, 27), and we have shown that the activation of phagocytosis by each lectin is related both to the carbohydrates exposed on the surface of the phagocytosed particle and to the sugar specificity of the lectin.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
West Indian leaf cockroaches (B. discoidalis) were kept in fiber-glass containers at 28°C, with a constant 12-h light/dark cycle. They were fed on dried cat food, banana skins, and apple cores, and given water ad libitum. Adult males and females were used for all of the experiments.
Bleeding of insects and preparation of monolayers
Cockroaches were cooled at -20°C for approximately 20 min and surface sterilized with 70% ethanol. Using a sterile 26G needle, 0.4 ml of anticoagulant (0.01 M EDTA (free acid), 0.1 M glucose, 0.062 M NaCl, 0.03 M trisodium citrate, 0.026 M citric acid (pH 4.6), 370 mOsm) was injected between the second and third abdominal sclerites. Insects were massaged gently to circulate anticoagulant before bleeding by piercing the arthrodial membrane of a posterior limb with a 21G needle, and 0.1 ml of hemolymph collected in a syringe containing 0.4 ml of anticoagulant. The contents of the syringe were mixed and ejected into a sterile 7-ml Bijou bottle containing 2 ml of Grace’s Insect Medium (GIM; Sigma, Poole, Dorset, U.K.). In cases in which incubation in PTU (phenylthiourea) was required to inhibit the prophenoloxidase (proPO) system, the contents of the syringe were ejected into 2 ml of GIM containing a few crystals of PTU. Bleeding of the insects was performed in a 4°C constant temperature room to prevent hemolymph coagulation.
For hemocyte monolayer preparation, sterile 5-mm diameter glass coverslips were placed into the wells of a sterile, 96-well, flat-bottom microtiter plate (Nunc, Roskilde, Denmark), and 50 µl of diluted hemolymph was pipetted into each well. The plate was then incubated at 30°C for 20 min to allow the cells to attach to the glass coverslip. To remove any unattached cells, the monolayers were washed three times with 100 µl of GIM.
Lectin labeling of hemocytes
A number of FITC-labeled lectins were used to probe the surface carbohydrates of the B. discoidalis hemocytes, namely Con A, WGA, HPA, and TPA (Sigma). All lectins were dissolved in GIM at a concentration of 100 µg/ml, aliquoted, and stored at -20°C.
Monolayers were overlaid with 50 µl of FITC-labeled lectin and incubated at 29°C for 15 min in the dark. Unbound lectin was gently washed off three times with 100 µl of GIM. The cells were fixed with GIM-buffered 4% formaldehyde for at least 30 min at 29°C. The fixative was washed off with GIM, and the 5-mm coverslips, containing the monolayers, were removed from the microtiter plate wells, placed on microscope slides, then mounted using Kaiser’s glycerol gelatin (50% glycerin, 8% gelatin, 0.01% merthiolate). At least 250 cells per monolayer were then examined under UV light using a Zeiss photomicroscope II, and equipped with a barrier and excitation filter set number 4877009 for FITC epifluorescence.
Lectins preincubated for 1 h with their respective inhibitory
sugars were used as negative controls. The inhibitory sugars used for
each lectin were as follows (the lectin that each sugar inhibits is
included in the parentheses): D-(+)-mannose
(0.2 M; Con A);
N,N',N"-triacetylchitotriose,
a trimer of N-acetyl-D-glucosamine (4.5 mM;
WGA); N-acetyl-D-galactosamine (1.5 mM; HPA);
and 
-D-(+)-fucose (0.2 M; TPA).
To investigate whether Con A was binding to the same ligands as BDL1, monolayers were incubated with 50 µl of either BDL1 or GIM for 30 min at 29°C. The cells were then washed five times with 50 µl of GIM, before incubating the cells in 50 µl of FITC-labeled Con A for 15 min at 29°C. The monolayers were washed five times with GIM and fixed and mounted as above.
Culture and labeling of yeast and bacteria
Baker’s yeast (Saccharomyces cerivisiae; Westmill Foods, Maidenhead, Berks, U.K.) was grown up in YM broth (Difco, E. Molesley, Surrey, U.K.), overnight at 25°C, in a shaking water bath. The cultured cells were heat killed in a boiling water bath for 1 h, then washed three times in pH 7.4 TBS/Ca2+ (20 mM Trizma base, 77 mM NaCl, 10 mM CaCl2) by centrifugation at 650 x g for 10 min. The washed cells were then labeled with FITC by the method of Rohloff et al. (28) and resuspended at a concentration of 1 x 107 cells/ml in GIM.
E. coli K12 and Bacillus cereus PC2599 were treated in a similar manner to the yeast cells, except they were grown in nutrient broth at 37°C and 25°C, respectively. The resultant cultures were then processed in the same way as the yeast, except that they were centrifuged at 2000 x g.
Preparation of the B. discoidalis lectins
The endogenous lectins BDL1, BDL2, and BDL3 were purified from the serum of adult cockroaches by a combination of ammonium sulfate precipitation, DEAE-cellulose cation-exchange chromatography, and affinity chromatography, as described in Chen et al. (22). GSL was purified by a combination of gel filtration and blue-Sepharose chromatography, according to the method of Chen (23). The purified lectins were dialyzed against GIM for use in the phagocytosis assays. The BDL1 used for the dose-dependency curves was purified by the following immunoaffinity method, which yields much greater volumes of almost pure lectin than the method of Chen et al. (22), with only one major contaminating protein.
Mouse anti-BDL1 mAb 6B4 was purified from 500 ml of hybridoma culture supernatant using a protein G-Sepharose 4B column (Sigma) (29). Fractions containing pure Ab were pooled (total of 12 mg Ab), concentrated down to 2 ml, and washed with carbonate buffer (pH 8) (0.1 M sodium carbonate in 0.5 M NaCl), using a Centriprep-30 concentrator. The concentrated Ab was then coupled to 0.5 g (dry weight) CNBr-activated Sepharose (Sigma), according to the manufacturer’s instructions, packed into a polypropylene minicolumn (Pharmacia, Piscataway, NJ), and equilibrated with TBS (pH 7.4).
A 2-ml aliquot of a 30–50% ammonium sulfate fraction of B. discoidalis serum was applied to the column and allowed to incubate for 1 h at 4°C. The throughflow was collected and reapplied to the column. This was repeated an additional two times. The column was then washed thoroughly, to remove all nonspecifically bound protein, using a stepped salt gradient starting with TBS (pH 7.4) alone, and increasing to 2 M NaCl in 0.5 M increments, then returning to TBS (pH 7.4) alone.
Bound protein was eluted with 10 ml of 0.1 M glycine (pH 2.5), and fractions were collected in 0.5-ml aliquots, in Eppendorf tubes containing 100 µl of 1 M Tris/HCl (pH 8). Protein-containing fractions (determined by absorbance at 280 nm) were tested for purity on SDS-PAGE, and the purest fractions were pooled, washed with GIM, and concentrated using a Centriprep-30 concentrator.
The activities of purified BDL1, BDL2, and BDL3 were determined by their ability to agglutinate rabbit erythrocytes, as described by Chen et al. (22), or, for GSL, the agglutination of yeast (23), and their concentrations were adjusted to 17,000 U/ml for BDL1, 5000 U/ml for BDL2, 10,000 U/ml for BDL3, and 760 U/ml for GSL, in which 1 U equals an agglutination titer of 1/mg of purified lectin.
Opsonization of yeast and bacteria
FITC-labeled bacteria and yeast suspensions (50 µl used per monolayer) were pelleted at 10,000 x g for 20 s. The supernatants were removed, and 50 µl of lectin were added with gentle agitation to resuspend the pellets, and incubated at 29°C for 1 h in the dark. The cells were pelleted by centrifugation, washed twice in 50 µl of GIM to remove any unbound lectin, and finally resuspended in GIM (50 µl). Inhibited lectin controls were prepared, as above, except the lectins were preincubated in their respective inhibitory sugars for 1 h at 29°C before addition to the bacteria or yeast. The inhibitory sugars used were: 0.2 M mannose for BDL1 and Con A; 0.2 M GlcNAc for BDL2; 1 mg/ml GalNAc for BDL3 and HPA; 1% laminarin for GSL; and 0.2 M fucose for TPA. BSA (100 µg/ml) was used as a proteinaceous nonlectin control.
Phagocytosis experiments
The method described below is a modification of the technique described by Rohloff et al. (28). To each hemocyte monolayer, 50 µl of opsonized bacteria or yeast (1 x 107 cells/ml), or 50 µl bacteria or yeast treated with BSA or GIM alone, were added, and incubated at 29°C. After 1 h, the monolayers were washed five times with 100 µl GIM in each well, to eliminate any noninternalized particles. Monolayers were then overlaid with 50 µl of 0.2% trypan blue, and incubated in the dark for 5 min at 29°C. All traces of trypan blue were washed away with GIM, and the cells were fixed as above for the lectin-labeling experiments. At least 250 cells per monolayer were then examined under UV light for phagocytosed bacteria or yeast using a Zeiss photomicroscope II, as above.
Treatment of monolayers with laminarin
To demonstrate the effect of the activation of the proPO system on phagocytosis, monolayers were preincubated with laminarin, a known activator of proPO. Since laminarin, a ß-1,3-glucan, is also an inhibitor of GSL receptor recognition, the proPO system was activated before the addition of the test particles. Adult cockroaches were bled, as described previously, except the contents of the syringe were ejected into a Bijou bottle containing 2 ml of a GIM-buffered solution of 1% (w/v) laminarin. Monolayers were prepared immediately from the resulting cell suspension, and the phagocytosis assay was performed as described above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The original method of purifying BDL1 by Chen et al. (22) yields approximately 100 µg of pure protein from 10 ml of serum. In comparison, the immunoaffinity method, described in this work, produces 100 µg of protein with a similar sp. act. to that purified by Chen et al. (22), but from only 1 ml of serum. The purified BDL1 is, however, not totally pure, as it is always copurified with a protein that has a mass of approximately 40 kDa under both reducing and nonreducing conditions in SDS-PAGE. Since the immunoaffinity-purified lectin and the pure lectin, when used at comparable sp. act., induced phagocytosis by the same amount, it was concluded that the contaminating protein was not contributing to the observed effects, and therefore the immunoaffinity-purified lectin was used in a limited number of experiments.
Lectin labeling of hemocytes
Con A, HPA, and WGA stained 100% of plasmatocytes from adult
B. discoidalis, with Con A giving the most intense staining
and WGA the weakest (Table I
). Only Con A
was observed to also bind to the granular cells. TPA did not bind to
any of the cell types. Lectin binding was completely inhibited by
incubating the lectins with their inhibitory sugars before their
addition to the monolayers. There was no significant difference in the
pattern of labeling between fixed and nonfixed hemocytes.
|
Phagocytosis of yeast
Nonopsonized yeast, incubated in GIM alone or in 100 µg/ml BSA,
was phagocytosed by 6.56 ± 0.59% and 5.87 ± 0.56% of
plasmatocytes, respectively (Fig. 1a). Granular cells did not
exhibit any observable phagocytosis. This basal, nonspecific
phagocytosis was increased to over 11% (Fig. 1a) when the
monolayers were incubated with laminarin, a known activator of the
proPO cascade, before the addition of yeast. This increase in
phagocytosis due to laminarin was completely inhibitable by PTU (Fig. 1a), an inhibitor of phenoloxidase (30). Regarding
phagocytosis of yeast in the presence of the endogenous lectins BDL1,
BDL2, BDL3, and GSL, a fivefold increase in internalization, from
6.56 ± 0.56% to 31.54 ± 2.03%, was observed when yeast
was opsonized with BDL1 (17,000 U/ml; Fig. 1a). This effect
was completely inhibited by mannose (Fig. 1a), and was dose
dependent (Fig. 2a), reaching
a maximum level of phagocytosis at a concentration of 13,000 U/ml.
Incubation of the yeast with GSL also induced a significant increase in
phagocytosis from 6.22 ± 1.48% to 11.86 ± 1.02%, which
was inhibited by preincubation of the lectin with laminarin (Fig. 1a). In contrast, pretreatment of yeast by either BDL2 or
BDL3 had no effect on phagocytosis (Fig. 1a).
|
|
a), incubation of yeast with
Con A caused a dose-dependent (Fig. 4a), threefold increase in
phagocytosis from 6.56 ± 0.59% to 19.13 ± 0.97%, which
was inhibitable by 0.2 M mannose. WGA also induced a significant
enhancement in phagocytosis from 6.56 ± 0.59% to 11.86 ±
1.02%, which was approximately the same level as that observed with
laminarin-incubated monolayers (Fig. 1
a). HPA and TPA had no
effect on the phagocytosis of yeast. The effects of both the endogenous
and exogenous lectins on the phagocytosis of yeast are summarized in
Table II.
|
|
|
The basal level of phagocytosis of E. coli in GIM
(5.36 ± 0.78%) and BSA (6.65 ± 1.86%), and the activation
due to laminarin (10.10 ± 2.25%; Fig. 1b) were
similar to those observed with yeast. The endogenous lectins BDL1,
BDL2, and BDL3 all induced significant increases in the phagocytosis of
E. coli from 4.80 ± 0.96% to 26.05 ± 2.39%,
15.02 ± 2.96%, and 35.04 ± 9.01%, respectively. All of
the increases were inhibitable by their respective sugar ligands,
namely mannose (BDL1), GlcNAc (BDL2), and GalNAc (BDL3). The
enhancement of phagocytosis of E. coli by BDL1 was dose
dependent (Fig. 2a), reaching a maximum level of
phagocytosis at approximately 9000 U/ml. BDL2 and BDL3 were not tested
for dose dependency due to inadequate supplies of purified lectin. In
contrast to the other endogenous lectins, preincubation of E.
coli with GSL (Fig. 1b) resulted in no increase in the
internalization of E. coli. When BDL1 (4500 U/ml) and BDL2
(5000 U/ml) were incubated together, the observed level of phagocytosis
(14.91 ± 5.88%) was not significantly different from that seen
with each lectin individually at comparable concentrations (17.09
± 3.73% and 15.02 ± 2.96%, respectively).
With the exogenous lectins (Fig. 3b), those with specificity
for mannose, GlcNAc, or GalNAc, i.e., Con A, WGA, and HPA, all
augmented the uptake of E. coli to a similar extent from
4.80 ± 0.96% for the GIM control to 29.66 ± 3.52%,
31.49 ± 6.79%, and 26.82 ± 6.81%, respectively. The
effects produced by the exogenous lectins were all dose dependent and
reached a maximum at approximately 80 µg/ml (Fig. 4b). In
the case of WGA, there also appeared to be a minimum activation
concentration (20 µg/ml) below which no significant increase occurred
(Fig. 4b). The effects of both the endogenous and exogenous
lectins on the phagocytosis of E. coli are summarized in
Table II.
Phagocytosis of B. cereus
The basal level of phagocytosis of B. cereus in GIM
alone (6.74 ± 1.72%), the level of phagocytosis in the presence
of BSA (8.10 ± 3.97%), and the level of phagocytosis due to the
activation of the proPO cascade with laminarin (9.60 ± 1.57%)
were similar to those observed with yeast and E. coli (Fig. 1c). In this case, preincubation of bacteria with BDL1,
BDL3, and GSL caused no activation of phagocytosis, whereas BDL2
induced a very high level at 49.25 ± 6.91%, which was almost
completely inhibitable by 1 mg/ml GlcNAc (Fig. 1c). The dose
dependency of BDL2 was sigmoidal in character (Fig. 2b),
with a minimum activation concentration of approximately 900 U/ml and a
phagocytic maximum at 5000 U/ml.
WGA and HPA both gave similar highly significant enhancements of
phagocytosis (28.09 ± 7.36% and 28.42 ± 10.08%,
respectively (Fig. 3c)), which were significantly inhibited
by their specific sugar ligands, while Con A and TPA had no effect on
the internalization of B. cereus. Both the enhancements due
to WGA and HPA were dose dependent and exhibited their maximum
augmentation of phagocytosis at approximately 80 µg/ml, with HPA also
showing a minimum activation concentration of 40 µg/ml (Fig. 4c). The effects of both endogenous and exogenous lectins on
the phagocytosis of B. cereus are summarized in Table II.
The presence of laminarin in monolayers treated with lectin-opsonized test particles had no effect on the lectin-induced enhancement of phagocytosis for any of the microorganisms tested.
Effect of PTU on the lectin-mediated enhancement of phagocytosis
PTU is a potent inhibitor of the proPO system, a melanization cascade that is involved in numerous immunologic reactions in invertebrates, including nonself recognition processes (31). In the present study, since it has been shown previously that lectins and the proPO system interact (24), PTU was used to determine whether proPO was involved in the lectin-mediated enhancement of phagocytosis.
The presence of PTU produced a total inhibition of the BDL1 and
GSL-induced phagocytosis of yeast (Fig. 5a), to levels (7.71 ±
2.53% and 5.47 ± 1.32%, respectively) that were not
significantly different from the basal level of phagocytosis in GIM
alone. In contrast, phagocytosis was completely unaffected by PTU when
BDL1, or BDL2, was incubated with E. coli (Fig. 5a). A different effect again was seen when BDL2 was
incubated with B. cereus, with the presence of PTU causing a
40% reduction in phagocytosis, from 48.11 ± 6.99% to 28.93
± 3.54% (Fig. 5a). This PTU-inhibited phagocytosis was
still significantly (p < 0.0001) higher than
the nonstimulated level due to GIM alone.
|
b). Only partial inhibition of the
lectin-enhanced phagocytosis was observed with all the other
lectin/test particle combinations that induced an increase in
phagocytosis. All of these partially inhibited levels of phagocytosis
were still significantly higher (p < 0.005)
than the respective basal levels of phagocytosis. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previously, reports of opsonic activities of insect lectins have demonstrated an enhanced phagocytic rate against Gram-negative bacteria (11, 14), fungal blastospores (9, 13), or erythrocytes (10). In the cockroach, B. discoidalis, four lectins, each exhibiting different carbohydrate specificities, have been isolated (22, 23). Two of these lectins, BDL1 and GSL, have been shown to share functional and structural similarities to known acute phase reactants.5 The present study demonstrates for the first time in an insect that multiple endogenous serum lectins are capable of potentiating the phagocytosis of a variety of very different microorganisms. The observed effects are inhibited significantly by the sugar ligands of the lectins and are, therefore, dependent on the carbohydrate recognition domain of the lectins. Thus, the lectins are probably acting as bridging molecules, by binding to the external polysaccharides of the bacteria and yeast and then to receptors on the surface of the plasmatocytes. This is substantiated by the use of the exogenous lectins, of similar sugar specificities to the B. discoidalis endogenous lectins, which produce analogous opsonic effects to those observed with the endogenous lectins, and which were also shown to be capable of binding to the surface of the plasmatocytes. The binding of the endogenous lectins to the hemocyte receptors is probably independent of the carbohydrate recognition domain, at least in the case of BDL1, since no observable reduction in the intensity of binding to the plasmatocytes by Con A was observed in the presence of BDL1.
To examine the role of the B. discoidalis lectins in the
immune system of B. discoidalis, it is essential to first
have a knowledge of the polysaccharides exposed on the surface of each
microorganism tested. For this reason, well-studied bacteria and yeast
were used. First, baker’s yeast, S. cerivisiae, is known to
have an abundant supply of polymannans on its cell surface (32), which
would provide numerous binding sites for the mannose-binding lectins,
BDL1 and Con A. Both of these lectins induced a high level of
phagocytosis of yeast. Another group of polysaccharides, abundant in
yeast cell walls, are the ß-1,3-glucans (27). GSL, as its name
suggests, binds exclusively to ß-1,3-glucans (23), and induced
phagocytosis to a level comparable with laminarin-activated cells. This
level is surprisingly small, considering the high agglutinating
activity of GSL toward yeast, and may suggest that the physiologic role
of GSL is more orientated toward the activation of the proPO system, or
some other unknown function, than directly toward nonself recognition.
Chitin, a polysaccharide composed of repeating units of GlcNAc and
N-acetylmuramic acid, is also present, in small amounts, on
the yeast cell surface (33). The presence of the GlcNAc residues should
provide binding opportunities for BDL2 and WGA. HPA should also bind to
the GlcNAc residues, since, although its highest specificity is for
GalNAc, it also has an affinity for GlcNAc (Table II). However, neither
BDL2 nor HPA had any effect on phagocytosis of yeast, and WGA, which
has a very high specificity for chitin, only induced an increase
comparable with that obtained by activation of the proPO cascade by
laminarin. These results seem to suggest that the chitin in yeast is
not present in sufficient quantities to induce a large phagocytic
response by B. discoidalis plasmatocytes.
E. coli is a Gram-negative bacterium, and, therefore, the
major constituent of its outer cell membrane is LPS (26). LPS is
basically composed of a membrane-bound lipid A moiety bound to a
polysaccharide chain of variable length and composition dependent on
the strain of bacterium. For these experiments, the laboratory strain
K12 was used, which has a relatively simple carbohydrate structure (26, 34) since it lacks the highly variable O-antigenic portion
of the polysaccharide chain. Of the lectins tested, only GSL and TPA
failed to significantly increase the phagocytosis of E.
coli, which, on examination of the structure of the K12 LPS, is
fairly predictable since neither ß-1,3-glucan nor fucose is present.
There are, however, numerous binding sites for BDL1 and Con A, since
both have activity toward glucose (Table II), while BDL2 and BDL3 can
bind to the galactose residues, and BDL2, WGA, and HPA can interact
with the terminal GlcNAc residue of E. coli K12 LPS (Table II). However, the structure of LPS alone does not explain the high
levels of phagocytosis obtained with either WGA or HPA, since the
terminal GlcNAc residue is not always present (26). The levels of
phagocytosis observed with WGA and HPA are, therefore, probably due to
binding of the enterobacterial common Ag that is found on the cell
surface of all members of the Enterobactericidae. This is a
7-kDa molecule composed of repeating units of GlcNAc and
N-acetyl-D-mannosaminuronic acid cross-linked to
palmitic acid residues. BDL2 is probably also capable of binding to the
GlcNAc residues in this molecule.
Finally, B. cereus, like all Gram-positive bacteria, has a
thick peptidoglycan layer covering the entire surface of the bacterium
(26). Peptidoglycan is comprised of repeating units of GlcNAc and
N-acetylmuramic acid linked by short polypeptide chains, and
from this it is obvious why only those lectins with a specificity
toward GlcNAc (BDL2, WGA, and HPA; Table II) were able to induce a
significant increase in the phagocytosis of B. cereus.
It has previously been shown that both endogenous B. discoidalis lectins and exogenous lectins are capable of activating the proPO system (24), a melanization cascade important in many invertebrate immune reactions. Also, phagocytosis of bacteria, by insect hemocytes, is enhanced by the activation of the proPO system by laminarin, a ß-1,3-glucan, as demonstrated both in the present study and in previous work (35). Therefore, to determine whether the lectin-induced enhancement of phagocytosis was mediated through the activation of the proPO system, or was operating through an independent mechanism, the proPO system was specifically inhibited by PTU.
Three different effects were observed with the various combinations of lectins and microorganisms when the proPO system was inhibited with PTU. First, as was the case with BDL1- and GSL-induced phagocytosis, the presence of PTU completely inhibited the lectin-induced increase in phagocytosis of yeast. Since both of these lectins have been demonstrated to cause an increase in the activation of the proPO system (24), it is probable that BDL1 and GSL are inducing the phagocytosis of yeast via the activation of the proPO system. Second, the opposite effect was observed, with PTU having no effect on the enhanced phagocytosis of E. coli in the presence of BDL1, BDL2, or Con A, or the WGA-induced phagocytosis of yeast, indicating that a proPO-independent activation pathway is involved. It may be that the lectin-mediated binding of E. coli to the surface of the hemocytes causes direct activation of the phagocytic machinery, possibly through a secondary messenger cascade, such as the binding of small GTPases, which are known to be involved in mammalian phagocytic mechanisms (36). Finally, an intermediate effect was observed with BDL2-opsonized B. cereus, and with the majority of the treatments opsonized with the exogenous lectins. In all of these cases, the level of enhancement of phagocytosis induced by the lectins was reduced by 40–50%, but remained at a level significantly higher than the basal level, indicating that both the proPO-dependent and proPO-independent mechanisms are involved in producing the lectin-induced level of phagocytosis.
The results presented in this work demonstrate that the multiple serum
lectins found in B. discoidalis, and also exogenous lectins,
are capable of acting as nonself recognition molecules for a variety of
potential pathogens, with each lectin potentiating a response with a
different set of microbes (summarized in Table II). Since insects lack
the Ab-based recognition system found in the vertebrates, the ability
of a panel of lectins to potentiate the uptake of a variety of
microorganisms is especially important in terms of invertebrate nonself
recognition. This study represents the first evidence in an
invertebrate that multiple, endogenous serum lectins, of different
carbohydrate specificities, are capable of acting as opsonins toward a
range of very different microorganisms. Such multiple lectins in
cockroaches and related insects not only obviate the need for an
Ab-based immune system, but also the presence of antibacterial
peptides, which, although present in many other insect species, are
apparently absent in cockroaches (unpublished observation). Once an
invading microorganism has been recognized and phagocytosed by the
numerous circulating hemocytes in B. discoidalis, it can be
dealt with by intracellular killing mechanisms such as those based on
superoxide generation (37).
In conclusion, this present study emphasizes the importance of innate immunity based on multiple lectins and activation of a complex, complement-like proPO cascade that is sufficient in cockroaches to obviate the need for circulating antibacterial peptides. The efficacy of the system is clearly illustrated by the huge success of these insects that apparently thrive in extremely inhospitable, microbial-rich environments.
|
Footnotes |
|---|
2 Current address: Department of Zoology, University of Aberdeen, Tillydrone Avenue, Old Aberdeen, Aberdeen, U.K., AB9 2TN.
3 Address correspondence and reprint requests to Dr. Norman A. Ratcliffe, Biomedical and Physiological Research Group, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea, U.K. SA2 8PP.
4 Abbreviations used in this paper: MBL, mannose-binding lectin; BDL, Blaberus discoidalis lectin; GalNAc, N-acetyl-D-galactosamine; GIM, Grace’s Insect Medium; GlcNAc, N-acetyl-D-glucosamine; GSL, ß-1,3-glucan-specific lectin; HPA, Helix pomatia agglutinin; proPO, prophenoloxidase; PTU, phenylthiourea; TPA, Tetragonolobus purpureas agglutinin; WGA, wheat germ agglutinin.
5 C. Chen, A. F. Rowley, R. P. Newton, and N. A. Ratcliffe. 1999. Identification, purification and properties of a B-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defense reactions. Submitted for publication.
Received for publication May 21, 1998. Accepted for publication October 9, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. K. D. Schnitger, H. Yassine, F. C. Kafatos, and M. A. Osta Two C-type Lectins Cooperate to Defend Anopheles gambiae against Gram-negative Bacteria J. Biol. Chem., June 26, 2009; 284(26): 17616 - 17624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhu, P. M. L. Ng, L. Wang, B. Ho, and J. L. Ding Diversity in lectins enables immune recognition and differentiation of wide spectrum of pathogens Int. Immunol., December 1, 2006; 18(12): 1671 - 1680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. O'Rourke, D. Baban, M. Demidova, R. Mott, and J. Hodgkin Genomic clusters, putative pathogen recognition molecules, and antimicrobial genes are induced by infection of C. elegans with M. nematophilum Genome Res., August 1, 2006; 16(8): 1005 - 1016. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Osta, G. K. Christophides, D. Vlachou, and F. C. Kafatos Innate immunity in the malaria vector Anopheles gambiae: comparative and functional genomics J. Exp. Biol., July 1, 2004; 207(15): 2551 - 2563. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. A. Whitten, I. F. Tew, B. L. Lee, and N. A. Ratcliffe A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in {beta}-1,3-Glucan Pattern Recognition and Cellular Encapsulation Reactions J. Immunol., February 15, 2004; 172(4): 2177 - 2185. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-Q. Yu and M. R. Kanost Immulectin-2, a Lipopolysaccharide-specific Lectin from an Insect, Manduca sexta, Is Induced in Response to Gram-negative Bacteria J. Biol. Chem., November 22, 2000; 275(48): 37373 - 37381. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
