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Effects of Low Molecular Constituents From Aloe Vera Gel On Oxidative Metabolism and Cytotoxic and Bactericidal Activities of Human Neutrophils
Effects of Low Molecular Constituents From Aloe Vera Gel On Oxidative Metabolism and Cytotoxic and Bactericidal Activities of Human Neutrophils
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Printed in Great Britain. © 1990 International Society for Immunopharmacology.
Abstract - - In traditional South-East Asian medicine the therapeutic value of the parenchymous leaf-gel of
Aloe vera for inflammatory-based diseases is well-reputed. The aim of this study is to investigate at which
level gel-constituents exert their activity. We show here that low -Mr constituents of an aqueous gel-extract
inhibit the release of reactive oxygen species (ROS) by PMA-stimulated human PMN. The compounds
inhibit the ROS-dependent extracellular effects of PMN such as lysis of red blood cells. The capacity of the
PMN to phagocytose and kill micro-organisms at the intracellular level is not affected. The inhibitory
activity of the low-Mr compounds is most pronounced in the PMA-induced ROS production, but is
significantly antagonized by the Ca-ionophore A23187. It is shown that the inhibitory effect of the low-Mr
compounds is the indirect result of the diminished availability of intracellular free Ca-ions.
In Third World countries and in many industrialized largely unknown. Recently, we have isolated poly-
countries medicines of plant-origin are commonly saccharides from the gel which activate alternative
used in basic health care. The preparation and use of pathway complement activity in vitro and have an
this type of medicines are largely based on empirism. adjuvants activity on specific antibody production
Despite the fact that a number of the preparations and T-cell activity in vivo ('t Hart, van den Berg,
have been used successfully for ages, only little is Kuis, van Dijk & Labadie, 1989). Moreover, we
known about the effects of plant constituents on the demonstrated that low-Mr substances from the gel
processes that cause the illness. inhibit the release of ROS from stimulated
A fairly well-documented preparation in tradition- neutrophils ('t Hart, van Enckevort, van Dijk, Zaat,
al medicine is the parenchymous leaf-gel o f A l o e vera de Silva & Labadie, 1988a),
(Grindlay & Reynolds, 1986). In the crude form or as The aim of this present study was to investigate the
aqueous extract the gel has been used to treat effects of low-Mr gel-constituents on the oxidative
different forms of inflammatory afflictions, ranging metabolism of neutrophils in more detail. Because
from infected wounds (local) to rheumatoid arthritis ROS are involved in the cytotoxic activity (McCord &
(systemic). In animal models a beneficial effect of Wong 1978; Clark & Klebanoff, 1979; Weiss, 1980;
the gel has been demonstrated on infectious inflam- Vercelotti, van Asbeck & Jacob, 1985) of activated
mation (Solar, Zeller, Rasolofonirina, Coulanges, neutrophils and in the intraphagosomal killing of
Ralamboranto, Andriatsimahavandy, Rakotovao & ingested micro-organisms (McCord & Wong, 1978;
LeDeaut, 1982), rheumatoid arthritis (Saito, Johnston, Keele, Misra, Lehmeijer, Webb, Baehner &
Ishiguro, Imanishi & Suzuki, 1982) and X-radiation Rajagapolan, 1975) the effects of low-Mr gel-
dermatitis (Lusbaugh & Hale, 1953). The mechan- constituents on these biological functions of the
isms underlying the therapeutic effects of the gel are neutrophils were investigated as well.
tTo whom correspondence should be addressed at: Department of Immunology, TNO Primate Centre, P.O. Box 5815,
2280 HV Rijswijk, The Netherlands.
427
428 L. A. 'T HARTet al.
EXPERIMENTAL PROCEDURES Ltd) and diluted in HBSS, containing 0.1% gelatin
(HBSS-gel) to a concentration of 107 micro-
Preparation of the low-Mr fraction from the gel organisms/ml.
The leaves of Aloe vera plants (family Liliaceae)
were harvested in Sri Lanka. The mucilagenous Neutrophils
parenchymous tissue was excised from freshly cut Blood was obtained from healthy donors by
leaves, lyophillized and kept in sealed plastic bags venapuncture. Neutrophils were collected by
until use. To obtain an aqueous extract, 10 g of sedimentation on a plasmasteril gradient for 25 rain
lyophillized gel were swollen in 1 liter of saline at at 37°C. The PMN-rich layer was washed twice in
room temperature under gentle stirring. The 20 mM phosphate-buffered saline (PBS, pH 7.4).
yellowish extract was centrifuged for 15 min at Contaminating erythrocytes were then removed by
1000 × g. After decanting the supernatant the pellet lysis with water for 40 s followed by the addition of
was suspended in 500 ml of saline, and the extraction an equal volume of two-times concentrated PBS.
procedure was repeated. After a final extraction with The cells were washed twice with PBS and finally
250 ml of saline the non-soluble residue (80 mg) was suspended to a concentration of 107 PMN/ml
discarded. The pooled supernatants were dialysed HBSS-gel. In thus prepared suspensions more than
three times against 10 liters of demineralized (demi) 98% of the cells were viable neutrophils as
water. The non-dialysable fraction, containing determined by trypan blue exclusion.
polysaccharides not interfering with neutrophil
activity ('t Hart et al., 1988a), was also discarded. Serum
The pooled dialysates were lyophillized and the
Serum was prepared from veneous blood of
residue was extracted with excess ethylacetate to free
healthy AB donors, and stored at - 2 0 ° C .
gel-compounds from NaC1. Attempts to purify the
active compounds from this fraction failed
repeatedly, because isolated compounds were highly Measurement of Oz-consumption and the release of
unstable under normal laboratory conditions and 0 2 and Hz02 by neutrophils
therewith lost all activity. In semi-purified gel- Oz-consumption by resting and PMA-stimulated
extracts the inhibitory constituents remain active. neutrophils (100 ng PMA/ml) (Sigma Chem. Co., St
Therefore the ethylacetate extract from the dialysate, Louis, MO) was measured polarographically (van
containing about 0.5 mg gel-extract per miUiter, was Dissel, Olivier, Leijh & van Furth, 1986). Briefly,
used for the experiments. In this study two extracts 300 ~1 of a suspension of 5 × 106 PMN/ml
from the same charge of lyophillized gel were used. RPMI-1640 (Flow Lab. Ltd, Irvine, U.K.) were
Both extracts were compared by analytical thin layer added to a thermostated reaction vessel (37°C). The
chromatography. The activity of the extracts was Oz-tension in the medium was measured with a
determined by the concentration (v/v) giving 50% miniature pOz-electrode and recorded on a BD 41
inhibition of luminol-dependent chemiluminescence chart recorder (Kipp & Zonen, Delft, The Nether-
(CL,u,0 by STZ- (serum-treated zymosan) stimulated lands). The rate of Oz-consumption (nmol
neutrophils 0cs0). The Ic50 of the extract used for 02 × min ~') was calculated by measuring the slope
the experiments in Fig. 1 and Table 2 is 10% (v/v), of the Oz-tension graph over a 10 to 15 rain period.
and of the extract used for the other experiments The results are expressed as nmol/(5 × 106 x 5 min).
20% (v/v). The assay to measure Oz -release by neutrophils at
rest and after stimulation with 100 ng P M A / m l was
done as described (Babior, Kipnes & Curnutte,
Micro-organisms 1973). Neutrophils (106 cells/ml) were incubated
Two different micro-organisms were used in this with 1 mM cytochrome c (Type IV, Sigma) at 37°C.
study, which both are highly sensitive to ROS After a 15-min incubation the cells were centri-
toxicity: Staphylococcus (S.) aureus (type 42 D) was fuged at 600 × g for 10 min (4°C) and the absorb-
grown overnight in Nutrient broth No. 2 (Oxoid Ltd, ance at 550 nm was determined. In control incuba-
Basingstoke, U.K.) at 37°C, and Candida albicans tions neutrophils were omitted or the incubates
(UC 820) was cultured for 24 h at 37°C in Sabouraud contained 0.37 mg/ml superoxide dismutase (SOD,
dextrose broth (Difco Laboratories, Detroit, MI). Sigma). Oz--production was calculated from the
The suspensions of micro-organisms were centri- increase in A550.... using the extinction coefficient
fuged for 10 rain at 1500 × g, washed twice with of 2.9 × 10~ mM ~cm ~ (data from Sigma).
Hank's balanced salt solution (HBSS pH 7.4, Oxoid The results of these experiments are expressed as
Effects of Low Molecular Constituents from Aloe vera Gel on PMN 429
nmol/(106 cells x 15 min. H202-release by resting 4 min at 110 x g. The numbers of viable bacteria in
and PMA-stimulated neutrophils (100 ng PMA/ml) the supernates were determined microbiologically
was assessed by the peroxidase-dependent oxidation using diagnostic-sensitivity-test agar plates (Oxoid
of scopoletin (Root, Metcalf, Ostino & Chance, Ltd). Phagocytosis was expressed as the percentage
1975). The results are expressed in nmol/(106 cells decrease of the number of viable extracellular
x 5 min). micro-organisms.
For assessment of the intracellular killing capacity
of neutrophils 500/al of 107 PMN/ml were mixed
Chemiluminescence assay f o r R O S production with 500 tal of 107 opsonized S. aureus particles/ml
and incubated under rotation (4 rev./min) for 3 rain
Procedures for measuring luminol-dependent
at 37°C in HBSS-gel supplemented with 10°70 AB
chemiluminescence (CLlum) of stimulated neutrophils
serum. Non-ingested bacteria were removed by two
were described in detail previously ('t Hart et aL,
washes with ice-cold HBSS-gel using differential
1988a). Stimulants in this assay were zymosan,
centrifugation (4 min at 110 x g at 4°C). After this
opsonized by incubation for 30 min at 37°C with
procedure no decrease in the number of viable cells,
3 vol. pooled human AB serum (STZ), 10 ng/ml
as determined by trypan blue exclusion, was found.
PMA and 4 ~M of the calcium-ionophore A23187.
The neutrophils were suspended in HBSS-gel to a
Luminol and A23187 were from Sigma Chemical
concentration of 5 x 106/ml and again incubated
Company, St Louis MO, U.S.A.
under rotation at 37°C in the presence of 10070 AB
serum. At various time-intervals 50/A samples were
taken and after lysing the neutrophils in water
Cytochemical staining o f granulocytes f o r containing 0.0107o bovine serum albumin (BSA,
intracellular R O S Oxoid Ltd) the number of viable micro-organisms
Intracellular 02 and H202 concentrations in was determined microbiologically as beyond.
neutrophils were assessed semiquantatively after Intracellular killing is expressed as the percentage
specific cytochemical staining (Briggs, Karnovsky & decrease in the number of viable ingested bacteria.
Karnovsky, 1975; Briggs, Robinson, Karnovsky & The assay for phagocytosis and intracellular
Karnovsky, 1986). killing of C. albicans has been described in detail
elsewhere (Leijh, van den Barsselaar & van Furth,
1977). Yeast cells cannot be separated from neutro-
Cytotoxicity assay phils by differential centrifugation. Therefore, the
decrease in the number of free yeast cells in the
The 0 2 -dependent cytotoxicity of PMA-
suspension was counted in a Bfirker haemocytometer
stimulated neutrophils (20 ng PMA/ml) was
as a measure of phagocytosis. The decrease in the
measured by determining the specific release of
total number of viable C. albicans in the incubation
radioactivity from 5~Cr_labelled rabbit red blood cells
mixture was taken as a measure of intracellular
as described in detail elsewhere ('t Hart, van
killing.
Enckevort, van Kessel, van Dijk & Labadie, 1988b).
tion of 4 x 106cells per ml R P M I and kept on ice till l o w - M r gel-extract on the O2-consumption and the
further use. Immediately before the test, 0.5 ml of O2 and H202 release by resting and PMA-stimulated
the cell suspension was centrifuged. The pellet was neutrophils was investigated. It should be noted that
suspended in an equal volume of incubation buffer scavenging of Oz- by l o w - M r compounds is
(pH 7.4), consisting of 145 m M NaC1, 5 m M KC1, negligible ('t Hart et al., 1988a). The results in
1 m M NazHPO4, 1 m M CaClz, 0.5 m M MgSO4, Table la show that the l o w - M r fraction had a
5 m M glucose and 10 m M N a - H e p e s and trans- significant stimulatory effect on the consumption of
ferred to a 1 ml quartz cuvette. After adding 150/xg O2 by resting but not by PMA-stimulated
Con A (Calbiochem, American Hoechst Corp., neutrophils. The stimulated 02 consumption could
San Diego, CA) (Cohen, Chovaniec, Wilson & not be attributed to the oxidation of l o w - M r
Newburger, 1982) the increase in light emission constituents. The decrease of the 02 tension in
was recorded in a spectro-fluorometer (Aminco RPMI-1640 was 0.45 in the absence and 0.50 nmol
Bowman) (excitation wavelength: 3 4 0 n m ; light O2/min in the presence of 20% l o w - M r fraction. The
emission was recorded at 410 and 485 nm). l o w - M r fraction completely abolished the release of
02- by resting and PMA-stimulated neutropbils
(Table lb), while the H202-release by P M A -
Statistical analysis stimulated, but not by resting neutrophils was
Statistical analysis of the effects of the l o w - M r significantly reduced (Table lc).
fraction was done with Student's t-test. Differences In a separate experiment intracellular 02 and
were regarded as statistically significant when P was H202-production in PMA-stimulated neutrophils
<0.01. was visualized by cytochemical staining. F r o m the
intensity of the dye-precipitate, ROS concentrations
RESULTS can be assessed semiquantitatively. The results show
no difference in the staining intensities when
Effect o f low-Mr compounds from the gel-extract on stimulation occurred in the presence as compared to
the oxidative metabolism o f human neutrophils in the absence of the l o w - M r fraction.
The finding that l o w - M r compounds of Aloe vera
extract abolish the production of CL~umby stimulated Effect o f the low-Mr fraction on the microbicidal
neutrophils ('t H a r t et al., 1988a), points at a and cytotoxic function o f neutrophils
possible interference with the oxidative metabolism W h e n released into the pericellular space 02
of these cells. Therefore the effect of a mediates the lysis of 5~Cr-labelled R R B C , which is a
Effects of Low Molecular Constituents from Aloe vera Gel on PMN 431
measure o f the cytotoxic potential o f P M A - s t i m u - sensitive to ROS (Lehrer, 1970; Rodey, Park,
lated neutrophils ('t H a r t et al., 1988b). The results W i n d h o r s t & G o o d , 1969).
in Table 2 show that this activity o f P M A - s t i m u l a t e d
neutrophils was m a r k e d l y inhibited by the l o w - M r The possible site o f action o f l o w - M r constituents
fraction. In experimental conditions, ROS p r o d u c t i o n by
W h e n released in the p h a g o s o m a l c o m p a r t m e n t neutrophils can be induced with different stimulants,
ROS contribute to the killing o f ingested micro- such as opsonized particles, which bind to surface-
organisms. The results in Table 3 show that the receptors, the p h o r b o l ester P M A , which directly
l o w - M r fraction h a d no effect on the capacity o f activates the regulatory cellular enzyme P K C , and
neutrophils to p h a g o c y t o s e opsonized micro- the C a - i o n o p h o r e A23187, which inserts an artificial
organisms. This result is in agreement with previous Ca-channel. In a next set o f experiments the
findings on the phagocytosis o f radiolabelled and influence o f the l o w - M r fraction on CL~um) induced
opsonized S. aureus particles ('t H a r t et al., 1988a). with either o f these stimulants was tested. The
As is s h o w n in the same table the l o w - M r fraction chosen l o w - M r c o n c e n t r a t i o n gave rise to a b o u t 50°7o
h a d no effect on the killing o f S. aureus or inhibition o f STZ-induced CLum (Fig. 1). As is
C. albicans, a l t h o u g h b o t h m i c r o - o r g a n i s m s are s h o w n in the same figure P M A - i n d u c e d CLlum was
432 L. A. 'T HARTet al.
100 -
verify this hypothesis the influence of the low-Mr
fraction on the rise of the intracellular free Ca 2÷
8O
concentration in stimulated neutrophils was
E determined. The lectin Con A was chosen for
d stimulation because in our hands this gave a strong
Z 60 and synchronous fluorescence signal. The results in
0
I---
Fig. 2 show that the low-Mr fraction abolished the
Con A-induced increase of the intracellular free Ca 2'
112 4 0
Z concentration.
20
DISCUSSION
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