ARTICLE
INTRODUCTION
A proline-rich polypeptide (PRP) was isolated from ovine colostrum by
Janusz et al. [1, 2]. The polypeptide showed immunoregulatory properties
inducing maturation and differentiation of murine thymocytes, and affected
humoral and cellular immune responses, both in vivo and in vitro
[2-4]. PRP was able to reduce high, and to elevate low humoral immune
responses to SRBC in mice [3]. The net effect of PRP depended on the actual
state of the animals studied. PRP seems to restore balance in cellular
immune functions. PRP is not species-specific and is active in mice [3,
4], humans [5], and rats [6]. The molecular weight of PRP, determined
in SDS-PAGE, is about 6,000 [2], and it has been recently found that PRP
is a complex of proline-rich polypeptides of molecular weight up to 6,000
[Kruzel ML, Janusz M, Lisowski J, Fischleight RV, Georgiades JA: Towards
an understanding of the biological role of Colostrinin peptides submitted].
PRP contains a high proportion of proline residues (25%) and hydrophobic
amino acids (40%). An active nonapeptide fragment (NP): Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro
was isolated from the chymotryptic digestion products of PRP. It showed
in mice biological activity similar to the undigested PRP [2, 7]. Studies
on the structure-activity relationship showed that the C-terminal fragment
of NP, a hexapeptide (HP): Tyr-Val-Pro-Leu-Phe-Pro, also exhibited immunotropic
activity similar to PRP and NP [8].
It was also found that the PRP complex, and to a lesser degree NP, is
a modest cytokine inducer (IFN, TNF-alpha) in human whole blood cell cultures
[5]. During our studies involving the possibility of using PRP as treatment
for correcting immune disorders, we noticed that volunteers who received
PRP showed signs of improvement in mood and cognitive abilities [5]. The
immunoregulatory and psychoenhancing activity of PRP prompted us to investigate
whether the polypeptide complex could be used for the treatment of Alzheimer's
disease. In a double-blind, placebo-controlled study, we showed that PRP,
in the form of orally administered tablets called Colostrinin®,
improves the outcome of Alzheimer's disease patients [9]. The ability
of PRP to induce secretion of IFN correlates well with the observation
that this interleukin inhibits the formation of beta-amyloid deposits
and might have a beneficial effects on Alzheimer patients [10-12]. PRP
also has beneficial effects on cognitive functioning in aged rats [6].
In some immune processes NP and HP showed biological activity similar
to that of the PRP complex, e.g. effect on the humoral or cellular
immune responses or on the resistance of murine thymocytes to apoptosis
induced by hydrocortisone [2]. However, NP and HP were inactive or showed
significantly lower activity than PRP in the induction of cytokines in
human pe-ripheral blood leukocytes [5] or in murine resident peritoneal
cells [13]. Therefore, it was of interest to synthesize analogs of NP
or HP which might show enhanced cytokine-inducing ability. For this purpose,
NP and HP containing D-amino acids as N-terminal amino acids and covalent
linear oligomers built of repeating units of NP or HP were synthesized:
NP1: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro
NP2: (Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro)2
NP-D: D-Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro
HP1: Tyr-Val-Pro-Leu-Phe-Pro
HP2: (Tyr-Val-Pro-Leu-Phe-Pro)2
HP3: (Tyr-Val-Pro-Leu-Phe-Pro)3
HP4: (Tyr-Val-Pro-Leu-Phe-Pro)4
HP-D: D-Tyr-Val-Pro-Leu-Phe-Pro
The aim of the present study was to examine whether linear oligomers
and D-analogs of NP1 or HP1 could express improved
cytokine-inducing activity compared to their monomeric analogs and PRP.
The immunotropic activity of the peptides was evaluated by determination
of the induction of interferons (IFN), tumor necrosis factor-alpha (TNF-alpha),
interleukin-6 (IL-6), and interleukin-10 (IL-10) in cultures of human
whole blood cells.
MATERIALS AND METHODS
Reagents
A proline-rich polypeptide complex (PRP) was isolated from ovine colostrum
in accordance with the method of Janusz et al. [1]. RPMI medium
was obtained from the Laboratory of Biopreparations of the Institute of
Immunology and Experimental Therapy, Wroclaw, Poland. Tissue culture plates
were from Costar, USA. Leukoagglutinin (PHA), bacterial lipopolysaccharide
(LPS) from E. coli, thiazolyl blue (MTT), actinomycin D, L-glutamine,
sodium dodecyl sulfate (SDS), dimethylformamide (DMF) were obtained from
Sigma, USA. Antibodies against IL-6 and IL-10, and recombinant IL-6 and
IL-10 were purchased from PharMingen, USA. Polypeptides: NP, HP, and their
linear covalent oligomers, NP and HP with D-amino acids at their N-terminal
ends were synthesized by the solid-phase method using the Boc/Bzl procedure
[14]. The peptides obtained were homogeneous on thin layer chromatography
plates and their purity exceeded 95%, as judged by HPLC. They revealed
the expected amino acid composition and molecular weight (MS-ESI).
Mice
Six week-old C57/BL/6 mice were purchased from the animal farm of the
Institute of Immunology and Experimental Therapy, Wroclaw, Poland.
Cytokine induction in human whole blood cell
cultures
The experiments were performed according to the procedure described
by Inglot et al. [5]. Blood samples were collected into syringes
containing 10 U/ml of heparin. Within 2 hours after the collection, the
blood was diluted 10-fold with RPMI-1640 medium supplemented with 100
units/ml penicillin, 100 mug/ml streptomycin, and 0.5 mg/ml L-glutamine.
One ml portions of the cell suspensions were distributed in duplicates
or triplicates into 24-well flat-bottomed tissue culture plates. The inducers,
PRP and synthetic peptides, were added in volumes of 20 mul RPMI containing
1 mug, 10 mug, and 100 mug of the peptides. The reference of positive
inducers were: 2 mug/ml of PHA plus 2 mug/ml of LPS. The control wells
contained the culture medium only and were used to measure the spontaneous
production of cytokines (negative control). The plates were incubated
for 20 hours at 37° C in a 5% CO2 atmosphere. After the
incubation, plates were centrifuged at 1,000 rpm for 15 min at 4°
C. The supernatants were collected and used for the determination of cytokines.
The samples were stored for several weeks at - 20° C before the determination
of cytokines.
Determination of cytokines
Interferons (IFN) and tumor necrosis factor-alpha (TNF-alpha) concentrations
were measured using bioassays [5].
Interferon bioassay. IFN activity was measured in A549 cells (human
lung adenocarcinoma, ATCC CCL 185). The confluent monolayers of A549 cells
were prepared in 96-well microplates in Dulbecco's modified Eagle's minimum
essential medium (DMEM) with 10% calf serum, L-glutamine, and antibiotics.
IFN samples diluted on separate plates were added to the cell monolayers
and incubated at 37° C for 24 hours at 5% CO2 in humidified
air. The cells were then washed and challenged with encephalomyocarditis
virus (EMCV). The MTT method was used to determine the EMCV cytopathogenic
effect and cytotoxicity of the peptides. The end-point of IFN titration
in the MTT assay was taken as 25% reduction of cell killing in comparison
with control cells infected with 100 TCID50 (tissue culture
infective dose) of EMCV producing complete cytotoxicity. Laboratory standards
of IFN were included in all assays.
Tumor necrosis factor-alpha (TNF-alpha) bioassay. The cytotoxic activity
of TNF-alpha was measured in L929 cells. Samples serially diluted in culture
medium supplemented with actinomycin D (5 mug/ml) were added to the monolayer
of cells cultured in 96-well microplates. After incubation for 20 hours
at 37° C, the cytotoxic effect was determined. The highest dilution
causing the death of 50% of cells in the culture was defined as one unit
of TNF-alpha activity. The standard preparation of recombinant TNF-alpha
was used as a control.
IL-6 and IL-10 immunoassays. The interleukins were determined by microplate
ELISA, using commercially available antibodies and recombinant interleukins
from PharMingen according to the procedure recommended by the manufacturer.
Statistical analysis
Data are presented as means ± SD from at least 3 independent experiments
performed in duplicate or triplicate. Data comparison were made with Student's
t test for independent samples. Differences were considered significant
when p was < 0.05.
RESULTS AND DISCUSSION
The cytokine induction was determined under ex vivo stimulation
of human whole blood cell cultures with LPS and PHA, PRP, NP1
or HP1 and their analogs. This method was selected because
it offers the great advantage of reproducing the natural microenvironment
of immunocompetent cells and because it preserves the various intercellular
communications between the different blood cell populations [15]. Using
this method, it is possible to avoid cell activation related to isolation
procedures [15, 16]. Cytokine production in whole blood cells can be used
as an indicator of immune cellular status [17, 18]. Two types of cytokines
were measured, IFN and TNF-alpha, secreted by Th1 cells and involved in
the cell-mediated immunity, and IL-6 and IL-10, secreted by Th2 cells
associated with the humoral immune response [18].
In all experiments, the cytokine-inducing ability of the PRP complex
and of the other peptides was much lower than that of LPS-PHA. (Tables
1, 2, 3 and 4). However, PRP, at concentrations of 1-100 mug/ml, was
able to induce the secretion of all cytokines studied (Tables
1, 2, 3 and 4). Maximal activity was observed at 10 mug/ml, with the
exception of IL-6 induction which was the highest at 100 mug/ml (Tables
3 and 4).
The cytokine inducing activity of HP1 was similar to the
activity of NP1. This resembles to the results of studies on
the effect of NP1 and HP1 on the humoral and cellular
immune responses, where both peptides showed similar effects [8]. These
peptides were generally less effective in the induction of cytokines,
compared to PRP, and their oligomerization showed different effects, depending
on the cytokine induced.
In the case of induction of IFN, the activity of the peptides NP1,
HP1, and their oligomers at 1 mug/ml and 10 mug/ml concentrations
was lower compared to PRP, was similar to or even higher and at 100 mug/ml
than the activity of PRP at 100 mug/ml. Generally, oligomerization of
NP1 and HP1 did not increase, or even decreased,
their IFN-inducing activity especially at lower concentrations (Tables
1 and 2).
The TNF-alpha-inducing ability of NP1 and NP2
(Table 1) was observed at concentrations
of 100 mug/ml only, and the dimer NP2 had the same activity
as the monomeric NP1. Oligomers HP2, HP3,
and HP4 showed the same activity as the monomeric HP1
(Table 2). At concentrations
of 100 mug/ml, HP peptides were more active than PRP at the same concentration.
In the case of induction of IL-6, NP1 and NP2,
HP1, HP2, and HP4 showed lower activity
than PRP (Tables 3 and 4). The
most active peptide was the oligomer HP3, which activity was
comparable to PRP.
IL-10-inducing ability of NP1 and NP2 was lower
than that of PRP. Dimer (NP2), was less active than the monomer
NP1 (Table 3). Interesting
results were obtained in the case of HP1 and its oligomers
HP2- HP4 (Table
4). The peptides HP1 and HP2, at concentrations
of 1 and 10 mug/ml, were less active than PRP, and at a concentration
of 100 mug/ml, they showed similar activity to PRP at the same concentration.
However, peptide HP3, and especially HP4, at concentration
of 100 mug/ml, surpassed the activity of PRP and approached the activity
of the control inducers (LPS + PHA). In conclusion, trimerization or tetramerization
of HP1 markedly and selectively enhanced its IL-10-inducing
ability.
Replacement of N-terminal L-amino acids by D-amino
acids might increase the resistance of the peptides to aminopeptidases
and so prolong their half-life in the organism. Therefore, analogs of
the NP1 and HP1, containing D-amino acids (NP-D
and HP-D), were obtained and their activity was determined (Tables 1,
2, 3 and 4). It was found however, that their cytokine-inducing activity
was lower than that of NP1 and HP1. Introduction
of D-amino acids may cause a change in the conformation of NP1
and HP1 and decrease their biological activity or the N-terminal
amino acid residue may be essential for the activity.
The cytokine-inducing activity of the peptides studied indicates that
they are bioavailable. However, the mechanism of the importation of these
peptides into cells is, as yet, unresolved. Some of the peptides might
behave as ligands and start transmission of signals into cells by as yet
unidentified surface receptors, and activate the cells. On the other hand,
it was found in other laboratories, that hydrophobic peptides containing
proline residues, as in the case of PRP, NP, and HP, can freely penetrate
cell membranes, enter and activate the cells and affect their effector
functions [19-23]. The peptides can also be delivered into cells by peptide-transporting
proteins which occur in all organisms to optimize utilization of the universal
peptide pool [24, 25]. The internalized peptides can show free movement
to cytoplasmic target proteins [20]. There is the possibility of their
interaction with kinases, adapter proteins [26] and/or protein tyrosine
phosphatases [27] that can cause an enhancement or attenuation of signals,
and finally, the effector activity of cells. Interaction of PRP complex
peptides with other proteins can be facilitated by the presence of block
sequences of proline residues [1], which may be recognized by proteins
containing SH3 domains [28]. Pro-Leu motifs, present in PRP, NP and HP
may be recognized by proteins with WW domains [29]. The structure of the
peptides studied suggests that they can also pass through the nuclear
membrane and regulate expression of various genes [19].
CONCLUSION
In summary, the results obtained show that at higher concentrations (at
least 100 mug/ml), NP, HP, and their oligomers can generally replace the
PRP complex in the induction of cytokines in human whole blood cell cultures.
However, only the PRP complex showed a bell-shaped dose-response curve.
Such a dependence indicating a regulatory activity of PRP was also observed
in other assays, e.g. the effect of PRP on the release of antibodies
from spleen cells [3]. This could be due to the fact that PRP is a complex
of proline-rich polypeptides which could act in concert giving a resultant
effect. NP, HP, and their oligomers showed a linear dose-response dependence.
The results presented here suggest the possibility of using a particular
peptide, at a proper concentration, to induce a particular cytokine and
so modulate the immune response. This was particularly observed in the
case of Th2 cytokines, IL-6 and IL-10, where oligomers of HP were relatively
strong inducers of the secretion of these cytokines.
We can assume that PRP acts like a "super-cytokine", initiating the
cytokine cascade which help to keep the balance between the Th1 and Th2
cells responsible for the outcome of diverse immune processes [18, 30,
31]. Single cytokines can affect cells in a different way than a mixture
of cytokines. They can mutually enhance or supress their effects on cells
[30].
Recently, the involvement of the immune system in the pathogenesis of
Alzheimer's disease and the role of cytokines in the regulation of neurodegenerative
processes has been accepted [32-35]. The "super-cytokine"-like properties
of the PRP complex shown in this paper may shed some light on the mechanism
of the positive therapeutic effects of PRP in Alzheimer patients.
Acknowledgments. This work was supported by grant No. 6 PO4B
013 11 from the Polish Committee for Scientific Research.
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