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Peptides 26 (2005) 2188–2192
Studies on the mechanism of action of a proline-rich polypeptide
complex (PRP): Effect on the stage of cell differentiation
Adriana Kubisa, Ewa Marcinkowskaa, Maria Januszb,∗,J´
ozef Lisowskib
aInstitute of Biochemistry and Molecular Biology, Wrocław University, Poland
bDepartment of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,
12 R. Weigla, 53-114 Wrocław, Poland
Received 22 February 2005; received in revised form 6 April 2005; accepted 6 April 2005
Available online 17 May 2005
Abstract
A proline-rich polypeptide complex (PRP) with immunoregulatory and procognitive activities shows beneficial effects in Alzheimer’s
disease (AD). The mechanism of action of PRP in AD is not yet clarified. Here, we present results of the effect of PRP on Vitamin D3-induced
phenotypic (CD11b and CD14) and functional (phagocytic) differentiation/maturation of monocytes/macrophages using the premonocytic
HL-60 cell line as a model. This cell line can be induced to differentiate into monocyte/macrophage cells by incubation with Vitamin D3.
However, when Vitamin D3was applied together with PRP, a 30–40% inhibition of the expression of the differentiation markers and an
over-60% inhibition of phagocytic ability were observed. When PRP was administered to the cells after treatment with Vitamin D3,no
attenuation of the differentiation/maturation process of the HL-60 cells was observed. This indicates that PRP affects the early stages of
differentiation/maturation of these cells. Our results, therefore, suggest that PRP, which affects the differentiation/maturation processes of
cells of monocyte/macrophage lineage, may regulate in this way the inflammatory processes in which these cells participate.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Proline-rich polypeptide complex (PRP); HL-60 cells; Vitamin D3; Differentiation/maturation; Phagocytosis
1. Introduction
Proline-rich polypeptide (PRP) isolated from ovine
colostrum is a complex of peptides of various molecular
masses ranging from 500 to 3000Da. It contains a high
proportion of hydrophobic amino acids (40%) and proline
residues (25%). Most PRP peptides are derived from casein
or a hypothetical -casein homolog and annexin. Peptides
with no significant homology to any specific protein in the
currentGenBankdatabasehavealsobeenidentified [20]. PRP
has shown immunoregulatory properties, inducing the matu-
rationanddifferentiationofmurinethymocytes,andaffecting
humoral and cellular immune responses, both in vivo and in
vitro [18,40,44]. The polypeptide is not species specific and
is active in mice [40,44], humans [16], and rats [32]. The
∗Corresponding author. Tel.: +48 71 337 11 72; fax: +48 71 337 13 82.
E-mail address: janusz@iitd.pan.wroc.pl (M. Janusz).
net effect of PRP depends on the actual immune status of
the animals studied. PRP seems to restore balance in cellu-
lar immune functions. It was shown that PRP has beneficial
effects on the cognitive functioning of older rats [32] and
improves mood and cognitive abilities in humans [16]. The
orally administered tablet form called Colostrinin®, contain-
ing 100g of PRP, improves the outcome of Alzheimer’s
disease (AD) patients and prevents further deterioration of
patient health status [2,21,22].
The mechanism of action of PRP/Colostrinin®in AD pa-
tients has not been yet clarified. It is known that the im-
mune system plays an important role in the pathogenesis
and regulation of neurodegenerative processes, including AD
[3,7,9,26]. A very important role, both positive and nega-
tive, is played by microglial cells. These are macrophages
of the central nervous system, which can remove deposits
of amyloid beta-peptides (A) or damaged cells by phago-
cytosis [3,9,10]. On the other hand, permanent activation of
0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2005.04.001
A. Kubis et al. / Peptides 26 (2005) 2188–2192 2189
microglial cells by Aaggregates can induce disturbances
in phagocytosis, such as inhibition of the processing inside
cells, damage to neurons by accumulated peptides, and un-
regulated secretion of various effectors, e.g. reactive oxygen
species, nitric oxide, and cytokines [3,7,9,12,26,35]. There-
fore, any substance, which can regulate the differentiation
and activation of cells and modulate their function, may ex-
hibit a beneficial effect on AD [13,37]. PRP exerts regulatory
activity on the secretion of cytokines [42], nitric oxide, and
reactive oxygen species [4,5,28,41,43].
To gain further insight into the mechanism of action of
PRP, we decided to study the effect of PRP on the differenti-
ation and phagocytic activity of monocyte/macrophage cells.
As human microglial cells are not easily available, we used
the human promyelocytic leukemia HL-60 cell line. HL-60
cells provide a good in vitro model system for studying the
cellular and molecular events involved in the differentiation
of normal and leukemic cells to mature monocytes and for
studying phagocytic activity [8]. This cell line can be induced
to differentiate into monocyte/macrophage cells by incuba-
tion with Vitamin D3[30] in a process resembling the normal
maturation of monocytes in bone marrow [6].
In this paper, we present results on the effect of PRP on
Vitamin D3-induced phenotypic and functional differentia-
tion of HL-60 cells. We found that both the expressions of
phenotypic markers (CD11b, CD14) and functional features
(phagocytosis) of the differentiation of HL-60 cells were in-
hibited by PRP.
2. Materials and methods
2.1. Reagents
Proline-rich polypeptide complex (PRP) was prepared
from ovine colostrum according to the procedure of Janusz
et al. [17].1␣,25-Dihydroxyvitamin D3(Calcitriol) was ob-
tained from Infarm, Warsaw, Poland. RPMI-1640 was ob-
tained from the Laboratory of Bio-agents of the Institute of
Immunology and Experimental Therapy, Wroclaw, Poland.
Inactivated fetal calf serum (FCS) was from Gibco, USA,
and penicillin and streptomycin from Polfa, Poznan, Poland.
The FITC-conjugated monoclonal antibodies (moAbs) anti-
CD11b (clone 44), anti-CD14 (clone UHCM-1), and anti-
HLA-DR (clone HK14) as well as bovine serum albumin
(BSA) were obtained from Sigma, USA. Cell tissue plates
were purchased from Costar, USA.
2.2. Cell lines and culture conditions
The human promyelocytic leukemia HL-60 cell line was
obtained from the European Type Culture Collection by
courtesy of Professor Spik and Dr. Mazurier (Laboratory
of Biological Chemistry USTL, Lille, France). The HL-60
cells were propagated as a suspension culture in RPMI 1640
mediumsupplemented with 8% fetal calfserum, 100 units/ml
penicillin, and 100mg/ml streptomycin. The cells were kept
at standard cell culture conditions, i.e., in a humidified atmo-
sphere of 95% air and 5% CO2at 37 ◦C.
2.3. Cell differentiation
The cultured cells were seeded at a density of
1.75×105cells/ml of culture medium on 24-well plates to
a final volume of 2ml. The cells were exposed to Vitamin
D3in concentrations 10−8M for 96h. To check the effect
of PRP on cell differentiation, PRP in concentrations of 0.1,
1.0, 10.0, and 100.0g/ml of cell culture was added simul-
taneously with Vitamin D3during the differentiation process
(on the third day) or after differentiation (96h). As a control,
undifferentiated and PRP-untreated cells were used. After in-
cubation the cells were collected by centrifugation, washed
in phosphate-buffered saline, and both phenotypic and func-
tional differentiation were determined.
2.4. Cell marker expression
To determine the expressions of CD11b, CD14, and HLA-
DR by flow cytometry, 2.0 ×105cellsin40lofPBS supple-
mented with 0.1% BSA and 0.01% NaN3were mixed with
an appropriate volume of FITC-conjugated moAb solution
(pre-chilled to 4◦C) in the concentrations recommended by
the producer. The cells were incubated for 45min in an ice
bathandsubsequently washed twice with 500 l of PBS(sup-
plemented as above). As a negative control, a normal rabbit
IgG fraction was used. Cell-surface fluorescence was mea-
sured using a FACS Calibur flow cytometer (Becton Dick-
inson, San Jose, CA, USA). Damaged cells were labeled by
adding 5l of propidium iodide solution (25 l/ml) to each
test tube just before data acquisition. Data from damaged
cells were not analyzed. Data analysis was performed using
Becton Dickinson Cell Quest software.
2.5. Phagocytosis
To test phagocytic capacity, dried yeast Saccharomyces
cerevisiae werepreparedasdescribedbyMarcinkowskaetal.
[24]. The phagocytic ability of the cells was described as the
percentage of cells that had ingested yeast cells stained with
trypanblueduring3 h of incubation. At least 200 cells/sample
were counted.
3. Results
3.1. Expression of differentiation markers
To examine the effect of Vitamin D3on the expression
of the differentiation markers in our cell line, HL-60 cells
were cultured in the absence or in the presence of Vitamin
D3. As shown in Table 1, the expression of HLA-DR on
HL-60 cells was low and did not change in the presence of
2190 A. Kubis et al. / Peptides 26 (2005) 2188–2192
Table 1
The effect of Vitamin D3and PRP on the expression of differentiation mark-
ers on HL-60 cells
Marker −Vitamin D3,
−PRP +Vitamin D3,
−PRP +Vitamin D3,
+PRP
HLA-DR 3.30 3.28 n.d.
CD11b 3.06±0.25 13.65±2.4a9.76±0.75a
CD14 2.74±0.14 41.02±10.78a24.68±6.09a
Cells were cultured (96h) in the absence or in the presence of Vitamin D3
(1×10−8M) or PRP (10g/ml simultaneously applied with Vitamin D3).
2.0×105cellsinPBSsupplemented with 0.1% BSA weremixedwithFITC-
conjugatedmonoclonalantibodiesagainstHLA-DR,CD11b, or CD14. Cell-
surface fluorescence (mean channel of fluorescence) was measured using a
FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA).
Damaged cells were labeled with propidium iodide and not analyzed. Data
analysis was performed using Becton Dickinson Cell Quest Software. Re-
sults are expressed as means±S.D. from at least three independent deter-
minations; n.d.: not determined.
aStatistically significant (p<0.05, +Vitamin D3vs. −Vitamin D3and
+PRP vs. −PRP).
Vitamin D3. However, the low expressions of the CD11b
and CD14 monocyte/macrophage markers in the absence of
VitaminD3werestronglyincreased(especiallythatofCD14)
in the presence of Vitamin D3.
3.2. Effect of PRP on the expressions of CD11b and
CD14 differentiation markers induced by Vitamin D3
HL-60 cells were incubated with Vitamin D3for 96h in
the absence or in the presence of PRP. When PRP was added
simultaneously with Vitamin D3, it attenuated the expres-
sion of the differentiation markers, especially that of CD14,
on the HL-60 cells (Table 1;Fig. 1). As shown in Fig. 2,
PRP in the range of 10−5to 10−2g/ml, inhibited the ex-
pressions of CD11b and CD14 antigens on HL-60 cells in
a dose-dependent manner. However, in the case of CD11b a
regulatory biphasic effect was observed (Fig. 2). No effects
were found when PRP was administered to the cells 72 or
96h after the application of Vitamin D3(not shown). It is
worth mentioning here that PRP, at the concentrations used,
did not affect the viability of the HL-60 cells.
3.3. Effect of PRP on the induction of phagocytic
activity by Vitamin D3
Phagocyticabilitywasevaluatedbymeasuringthenumber
of cells which ingested yeast cells during 3 h of incubation at
37◦C. PRP alone did not induce phagocytic ability in HL-60
cells. As shown in Table 2, Vitamin D3strongly enhanced
the phagocytic activity of the HL-60 cell line (1.79% ver-
sus 54.6%). However, when HL-60 cells were preincubated
with Vitamin D3in the presence of PRP (1–100g/ml), a
significant, dose-independent inhibition (by about 60%) of
the phagocytic ability of HL-60 cells was observed. No in-
hibitory effects were observed in the presence of BSA and
Poly-Pro, used as controls.
Fig. 1. The effect of PRP on the expressions of CD11b and CD14 on HL-
60 cells exposed to Vitamin D3. HL-60 cells (1.25×105) untreated with
Vitamin D3(control), treated with 1×10−8M of Vitamin D3only (...), or
treated with Vitamin D3and 10 g/ml of PRP (—) were incubated for 96h at
37◦C. Cell-surface marker expressions were determined by flow cytometry
(FACS Calibur,BectonDickinson) using FITC-conjugated antibodies. Other
details as described under Table 1.
Fig. 2. The dose–response effect of PRP on the expression of differentiation
markers induced by Vitamin D3on HL-60 cells. Cells were incubated with
Vitamin D3(96h) in the absence or in the presence of PRP in concentra-
tions of 10−5to 102g/ml. The expressions of cell-surface markers were
determined by flow cytometry as described under Fig. 1. The mean canal of
fluorescence (MC) of cells treated with different concentration of PRP was
measured. MC of cells untreated with PRP was taken as 100%. () CD11b;
() CD14.
A. Kubis et al. / Peptides 26 (2005) 2188–2192 2191
Table 2
The effect of PRP on the phagocytic activity of HL-60 cells
Differentiation factor Percentage of phagocytic cells
(1) – 1.79 ±1.75
(2) Vitamin D354.6 ±8.36a
(3) PRP 1 g 2.3 ±0.94
(4) PRP 10 g 3.31 ±0.92
(5) PRP 100 g 2.16 ±1.25
(6) Vitamin D3+PRP 1g 19.7 ±6.97b
(7) Vitamin D3+PRP 10g 20.9 ±6.18b
(8) Vitamin D3+PRP 100g 21.5 ±5.19b
(9) Vitamin D3+BSA 10g 55.3 ±4.96
(10) Vitamin D3+Poly-Pro 10g 57.0 ±5.1
The percentage of phagocytic cells was determined as the number of cells,
which ingested yeast cells stained with trypan blue (at least 200 cells/sample
were counted). Results of the experiments are expressed as means±S.D.
from at least three independent determinations performed in duplicates or
triplicates.
aStatistically significant difference (p<0.05) between control and Vita-
min D3-treated cells (2 vs. 1).
bStatistically significant difference (p< 0.05) between cells untreated and
treated with PRP (6, 7, and 8 vs. 2).
4. Discussion
Phagocytosis plays an important role in the pathogenesis
ofAlzheimer’sdisease,bothpositive and negative.Microglial
cells can, by phagocytosis, remove deposits of amyloid-beta
peptides or damaged cells [1,34,39]. On the other hand, there
is also evidence that an intracellular accumulation of Amay
also be neurotoxic [27]. Microglial cells play an important
role in the central nervous system’s (CNS) immune responses
in Alzheimer’s disease [29]. They are similar to blood-borne
macrophages, which strongly suggests a bone-marrow ori-
gin of microglia and supports their identification as tissue
macrophages of the CNS [10]. Microglia are important in
the first line of defense of the CNS due to their ability to
phagocytose and process foreign antigen. Some of them are
activated resting microglial cells. More are monocytes that
have migrated through the walls of small vessels into the
central nervous tissue [29]. Chronic activation of microglial
cells in AD may result in an inflammatory response, distur-
bance of phagocytosis, deregulation of cytokine cross-talk,
overproduction of nitric oxide and reactive oxygen species,
and further deterioration of AD health status. Therefore, any
substance,which can regulate the differentiation and function
of cells, might exhibit beneficial effects in AD.
In this paper, we presented the results of studies on the
effect of PRP on the differentiation/maturation of mono-
cytes/macrophagesusingasamodel the premonocytic HL-60
cell line, which expresses a Vitamin D3receptor [15,31]. HL-
60 can be induced to differentiate into monocyte/macrophage
cells by incubation with Vitamin D3 in a process resembling
the normal maturation of monocytes in bone marrow [6,30].
To follow the differentiation of these cells, we measured
the expressions of CD11b and CD14, differentiation markers
characteristic of monocytes/macrophages and the phagocytic
ability of the cells.
HL-60 showed no phagocytic activity and only low ex-
pressions of CD11b and CD14. Incubation of these cells with
Vitamin D3strongly induced the expression of their differ-
entiation markers, which paralleled their phagocytic ability.
PRP showed an inhibitory effect on the differentiation of HL-
60 cells, reflected in a decrease in the expression of the dif-
ferentiation markers and their ability to phagocytose induced
by Vitamin D3. The effect was observed only when PRP was
applied concomitantly with Vitamin D3. This suggests that
PRP affected the early stages of differentiation of the HL-60
cells and that the presence of PRP did not exert an influence
on differentiated (Vitamin D3-treated) cells. This indicated
that the inhibitory effect of PRP on cell differentiation was
dependent on the initial stage of differentiation/maturation of
the cells used in our experiments.
There are several possible reasons for the inhibition of dif-
ferentiation/maturation of HL-60 cells by PRP. It is known
that PI-3 kinase [25] and mitogen-activated protein kinase
(MAPK)[15,19,23,33,38] signaling pathways play an impor-
tantroleinthedifferentiationofHL-60cellswithVitaminD3.
Therefore, we may assume that PRP might influence these
pathways. Hydrophobic peptides containing poly-proline se-
quences [11,14], as in the case of PRP peptides, can freely
penetrate cell membranes, enter the cells, and may be recog-
nized by proteins containing SH3 domains [36], which play
an important role in the activation/inactivation of cellular ki-
nases active in signal transmission.
The results presented in this paper indicate that PRP might
affect the differentiation/maturation processes of cells of
monocyte/macrophage lineage and thereby, regulate inflam-
matory processes.
Acknowledgment
This work was supported by grant No. 3 PO5A07525 from
the Polish Committee for Scientific Research (KBN).
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