(Received for publication, January 18, 1996; and in revised form, March 15, 1996 )
From the
Hemolymph of fifth instar Manduca sexta larvae collected under non-sterile conditions exhibited the presence of a novel high molecular weight protein complex, which was absent from the hemolymph collected aseptically. The high molecular weight complex consisted of, at least, prophenol oxidase, phenol oxidase, and an interleukin 1-like molecule, thereby demonstrating the generation of this complex as a consequence of a host defense response. While the native phenol oxidase and the interleukin 1-like molecule possessed molecular weights of about 80,000 and 17,000, respectively, the complex had a molecular weight of about 400,000. Apart from prophenol oxidase, phenol oxidase, and interleukin 1, dopachrome isomerase and other, as of yet unidentified, proteins may be part of the complex as judged by the presence of additional bands observed during SDS-polyacrylamide gel electrophoresis. The significance of the assembly of this defense complex for insect host defense strategies is discussed.
Invertebrate organisms have developed a plethora of defense reactions to fight invading parasites and pathogens. Insects and other arthropods in general do not possess immunoglobulins found in higher animals, although proteins containing immunoglobulin-like domains have been identified in insects(1) . Synthesis and secretion of antibacterial and antifungal proteins, agglutination and nodule formation, encapsulation of foreign objects, and phagocytosis are a few of the defense mechanisms insects use to protect themselves against infectious agents(2, 3, 4, 5, 6) . During defense reactions, invariably the foreign organisms are found to be encapsulated and melanized(5, 6, 7, 8, 9, 10, 11, 12) . Therefore, melanin and the enzyme responsible for its biosynthesis (i.e. phenol oxidase) are considered an integral part of insect host defense reactions. Phenol oxidase is present throughout the body of insects including the open circulatory system of hemolymph (5, 6, 7, 8) . Active phenol oxidase is deleterious as it can catalyze the oxidative polymerization of phenols and catechols, but in doing so, it can also polymerize proteins and other macromolecules, posing a potential threat to the host(7, 8) . Hence phenol oxidase is preserved as an inactive proenzyme form (prophenol oxidase) and is specifically activated proteolytically when needed(5, 6, 7, 8) . We have been examining the control mechanisms associated with the prophenol oxidase cascade and discovered: (a) a protease inhibitor controlling the prophenol oxidase cascade(9) , (b) quinone isomerase that inactivates deleterious 4-alkylquinones that are formed during phenol oxidase action(10) , and (c) phospholipid-mediated activation of prophenol oxidase(11) .
In insects and other arthropods, phenol oxidase also forms the terminal components of a cascade of reactions resembling the complement system of vertebrates(5, 6, 7, 8, 11, 12) . As a consequence, during invasion by a foreign organism, which is too large to be phagocytosed, inactive prophenol oxidase found in the hemolymph gets activated and participates in the encapsulation and melanization of the intruder. Thus damage that can be caused by the foreign object is limited by physical isolation. In addition, quinonoid compounds, being highly cytotoxic(13, 14) , could participate in the killing process, but this hypothesis requires verification(7) .
Numerous studies have been carried out on the activation of prophenol oxidase in insects(8) . Some authors have suggested that it is part of a recognition system(6) . This hypothesis needs to be proven although there is no doubt that the generation of phenol oxidase is a consequence of final reactions triggered by the host defense system.
Cytokines are polypeptide mediators released by a variety of activated immune and nonimmune cells and are major immunoregulatory proteins in mammals(15) . Equally important are the effects of cytokines as mediators of nonspecific host defense mechanisms(15, 16) . Biochemical characterization and studies of the molecular biology of cytokines from a number of vertebrate and invertebrate species have revealed basic similarities in the structure and biological properties of these important host defense molecules(15, 16, 17, 18) . Isolation of cytokine-like molecules from several invertebrate phyla attests to their importance to animal host defense systems(15, 18) .
The cytokine interleukin
(IL)()-1 was one of the first cytokines
described(16, 17) . It is an 18-kDa protein that has
numerous host defense-related properties. The pivotal role of IL-1 in
the immune response is demonstrated by its varied activities. Among its
myriad activities, IL-1 elicits the release of neutrophils from the
bone marrow, increases the expression of IL-2 receptors on lymphocytes,
and stimulates T cell proliferation(16) . In addition, IL-1
functions as a critical molecule in innate host defense responses.
Thus, IL-1 stimulates acute phase protein release from the liver,
causes the extravasation of neutrophils, increases vascular
permeability, and regulates the production of
fever(16, 17) . We have characterized IL-1 and other
cytokine-like molecules from invertebrates and studied the release of
these critical mediators by invertebrate coelomocytes (15, 18) .
In this communication we describe the assembly of a novel defense complex consisting of phenol oxidase, prophenol oxidase, and a hitherto unidentified IL-1-like molecule from the hemolymph of Manduca sexta.
SDS-PAGE was performed exactly as described previously(18) .
For Western analysis, gels were electroblotted in Towbin's
transfer buffer onto 0.45-µm nitrocellulose membranes (MSI,
Westboro, MA) at 200 mA for 2 h(24) . The blots were blocked in
Tris-buffered saline (TBS; 20 mM Tris-HCl, 150 mM NaCl (pH 7.5)) containing 0.1% Tween 20 (TBST) and 5% (w/v) nonfat
dry milk. They were washed in TBST, incubated for 2 h in primary
antibody (a 1:1,000 dilution of both rabbit anti-IL-1 and rabbit
anti-IL-1
(Dr. P. Conlon of Immunex)), washed, and incubated for 1
h in secondary antibody (2 µg of alkaline phosphatase conjugate of
goat anti-rabbit IgG/ml). After washing in TBS, the blots were reacted
with substrate (p-nitroblue tetrazolium chloride and
5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt).
Fig. 1shows the elution profile of M. sexta larval hemolymph proteins collected under both non-sterile and
sterile conditions. From the figure it is evident that a high molecular
weight (M) protein (complex) is present in the
hemolymph collected under non-sterile conditions (Fig. 1a, fractions 64-73), which is noticeably
absent in the sterile hemolymph (Fig. 1b). Phenol
oxidase is known to be activated during non-sterile conditions in M. sexta(20) as well as most other
arthropods(8) . Therefore, we reasoned that the new peak is due
to active phenol oxidase. Accordingly, activity measurements confirmed
the presence of phenol oxidase in this peak. The native M
of prophenol oxidase is about 80,000 (21) . Since the active enzyme is produced by the proteolytic
severing of a peptide from the proenzyme(5, 21) , one
would naturally expect the active enzyme to possess a M
of less than 80,000. In contrast, the approximate M
of the phenol oxidase generated under
non-sterile conditions was determined to be about 400,000 (Fig. 1a), indicating the self-polymerization of phenol
oxidase and/or complex formation with other hemolymph proteins.
Figure 1:
Characterization of
the high M complex. Concentrated hemolymph from
the fifth instar larvae of M. sexta was chromatographed on a
Sepharose 6B column. Column fractions were tested for prophenol oxidase
(
) or phenol oxidase (
) activity. a, unsterile
hemolymph; b, sterile hemolymph.
, A
. The positions of the M
markers are shown across the top of the
figure.
Since phenol oxidase is generated from prophenol oxidase, we looked
specifically for this precursor in the complex. Accordingly, the large M complex also contained prophenol oxidase (Fig. 1a). The total phenol oxidase activity in the
peak dramatically increased 2-3-fold when the proenzyme was
activated with cetylpyridinium chloride. In order to confirm the
presence of proenzyme, the putative complex was subjected to native gel
electrophoresis on 4-15% gradient gels and stained individually
for both phenol oxidase and prophenol oxidase. The results shown in Fig. 2confirm that both active phenol oxidase and inactive
prophenol oxidase are present in the large M
peak.
Figure 2:
Native and PAGE analysis of the Manduca high M complex. Pooled fractions
64-73 from the Sepharose 6B column (see Fig. 1a)
were concentrated by ultrafiltration and run on a 4-20% gradient
native PAGE gel. The gel was stained individually for both phenol
oxidase (lane 1) and prophenol oxidase (lane 2). A
Western blot (lane 3) was performed using a mixture of rabbit
antisera to human rhIL-1
and -
. Using SDS-PAGE, the peak
contained only two bands when stained with Coomassie
Blue.
Melanin production in animals is generally enhanced by the enzyme,
dopachrome isomerase(25) . In insects, this enzyme catalyzes
the conversion of L-dopachrome to 5,6-dihydroxyindole and
provides the indole for further oxidation by tyrosinase. Therefore, the
presence of dopachrome isomerase usually enhances melanin production.
Repeated attempts to detect this enzyme in the complex did not yield
consistent results. In only 10% of our experiments could we observe the
association of dopachrome isomerase with the large M peak (data not shown). Therefore, the association of dopachrome
isomerase with the complex is unclear at this time.
We then focused
our attention on the characterization of an IL-1-like molecule. During
a comparative search for IL-1 molecules in different arthropods, we
analyzed the hemolymph of M. sexta for IL-1-like activity. As
shown in Fig. 3, the presence of an IL-1-like protein with a M close to that reported in both vertebrates and
invertebrates (
20 kDa)(15, 16, 17, 18) could be readily demonstrated in the hemolymph of M.
sexta (fractions 173-186). (
)But what is more
intriguing is the association of this protein with the large M
complex, as seen when the hemolymph was
collected under non-sterile conditions (Fig. 3, fractions
64-73). The result indicates that the complex formation does not
interfere with IL-1-like activity and that the complex may be a carrier
for IL-1 in hemolymph. No IL-1-like activity was detected in the high M
column fractions from the hemolymph collected
under sterile conditions (data not shown).
Figure 3:
Presence of IL-1-like activity in the high M complex. Concentrated hemolymph from the fifth
instar larvae of M. sexta was chromatographed on a Sepharose
6B column (see Fig. 1a). Column fractions were tested
in the A375 assay. The data presented are for the 1:64 dilution.
,
IL-1 activity;
, A
. The positions of the M
markers are shown across the top of the
figure.
Further confirmation for
the presence of IL-1-like proteins in the complex comes from Western
blot analysis of the hemolymph proteins probed with antibodies to human
IL-1. As shown in Fig. 2, the high M complex peak readily cross-reacted with the antibodies to human
IL-1, confirming the presence of an IL-1-like protein in the complex.
To identify further the participation of an IL-1-like protein in the
complex, the antibodies to human IL-1 and -
were used in the
bioassay for IL-1. The high M
peak (Fig. 3,
fractions 64-73) was incubated with antibodies for 30 min before
the addition of the cells. As can be seen in Table 1the
antibodies to human IL-1, alone or in combination, inhibited the insect
IL-1-like activity. As we have observed in previous studies using human
antisera with invertebrate IL-1-like proteins, a maximum of
60%
inhibition in the A375 bioassay was observed(18) . Negative
controls (column buffer, fractions 4-14; and nonspecific
proteins, fractions 130-145) incubated with the antibodies had no
activity. In addition, a nonspecific control antiserum (rabbit antibody
to human IgG) had no effect when added to the high M
peak (data not shown).
Large M host
defense-related complexes have been described in mammalian
systems(26) . It usually entails the binding of cytokines by
the plasma protease inhibitor
-macroglobulin
(
-M)(27) . The binding is thought to be
mediated by an internal thiol ester bond(28) . The
cytokine
-M complexes have a relative M
of
400,000. Several cytokines have been
identified bound to
-M; these include IL-1, IL-2,
IL-6, tumor necrosis factor, transforming growth factor
, and
fibroblast growth factor(29) . It is thought the
cytokine
-M complexes are an important mechanism
for the modulation of cytokine activity(27) . It is interesting
to note that prophenol oxidase also contains a thiol ester
sequence(21) , similar to that found in
-M and
complement proteins (C3 and C4).
The function of this large M complex in Manduca is only speculative
at this time. The assembly of this complex in the activated hemolymph
attests to its host defense-related activity. When the prophenol
oxidase cascade is activated, the phenol oxidase should be deposited on
foreign (non-self) matter so that melanization can take
place(30) . The generation of toxic quinonoid products in the
circulatory system would be deleterious to the host. Therefore, the
open circulatory system of insects dictates localization of phenol
oxidase action at the desired sites only. One way this could be
achieved is by the generation of a complex consisting of, at least,
phenol oxidase with subsequent deposition on the foreign matter. The
recently identified thiol ester sequence of Manduca prophenol
oxidase (21) may be involved in covalent binding to cell
surfaces (i.e. the complex may possess characteristics to
allow covalent binding to the foreign object). In addition, the complex
may have effects on the distribution, availability, and clearance of
the IL-1-like molecule. This would direct the cytokines to the site of
infection rather than distributing them throughout the hemolymph. It
may also be involved with inhibiting the degradation of the cytokine(s)
as has been shown for cytokine
-M
complexes(27, 29) .
In conclusion, these data
indicate the formation of a high M complex during
an experimental insect host defense response. The complex is made up of
at least an IL-1-like molecule, prophenol oxidase, and phenol oxidase.
At this time, other components of this complex are being sought.
Further experiments on the physiological role as well as the
biochemical composition and metabolic fate of this novel complex are
ongoing in our laboratories.