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Production in Rat Macrophages1
*
Institute of Pharmacological Sciences, University of Milan, Milan, Italy;
Department of Neurosciences, University of Rome Tor Vergata, Rome, Italy; and
Institute of Pharmacology, University of Pavia, Pavia, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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from LPS-stimulated rat alveolar macrophages were
determined and correlated with a decrease in the level of RACK1, the
anchoring protein involved in protein kinase C translocation and
activation. Macrophages from aged rats produced 
50% less TNF-
than those from young rats. This effect was observed independently from
the concentration of LPS used and the time considered. The decrease
observed was associated with a defective PKC translocation, due to a
reduction in the expression of RACK1, whereas no differences were
detected in the expression of LPS receptor (CD14) or total PKC isoforms
( and ßII) in old and young rats. Use of RACK1
antisense oligonucleotide reduced the ability of young macrophages to
respond to LPS, further supporting the idea that a deficit in RACK1
contributes to the functional impairment in aged macrophages and that
age-induced macrophage immunodeficiencies are associated with
alteration in signal transduction pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF- is a potent paracrine and endocrine mediator of inflammatory
and immune functions. TNF- plays a crucial role in the development
of a protective response to bacterial (12) and viral
pathogens (13, 14). TNF- has been also implicated in
the mediation of a number of diseases including septic shock syndrome
(15), cachexia (16), and AIDS (17, 18) and in the pathogenesis of certain autoimmune diseases
(19). The primary sources of TNF- are activated
monocytes and macrophages (20), but it is also produced by
a number of additional cell types including mast cells
(21), granulocytes (22), B and T cells
(23, 24), keratinocytes (25), astrocytes
(26), etc.
Several studies have demonstrated that specific inhibitors of protein
kinase C (PKC)3 can
block the secretion of TNF- from LPS-stimulated macrophages,
suggesting that PKC activation plays a significant role in the
inflammatory response (27, 28, 29). PKC is a family of
phospholipid-dependent serine-threonine kinases involved in the signal
transduction of hormones, neurotransmitters, and cytokines
(30). Molecular cloning and biochemical studies
(31) have provided a basis for classifying the different
PKC isoenzymes into calcium-dependent (, ßI,
ßII, and 
) and calcium-independent (novel
PKC-
, -
, -
, -
, and -µ; atypical PKC-
, -
and -
)
species. The activation of PKC results in redistribution
(translocation) of the enzyme from cytosolic to membrane compartments
(31). Recently, a family of proteins that interact with
PKC has been described (32). These receptors for activated
C kinase (RACKs) are 30- to 36-kDa proteins located in various
subcellular compartments. The RACKs interact with PKC only in the
presence of kinase activators in a specific and saturable manner
(32, 33, 34). RACK1, a 36-kDa protein cloned from rat brain,
is the best characterized member of the RACK family (for a recent
review, see 35). Based on experiments with various PKC isoforms
and isoform-specific Abs, it has been demonstrated that RACK1
preferentially interacts in vivo with PKC-ß as compared with other
PKC isoforms. Studies in LPS-activated macrophages have indicated the
activation of several PKC isoforms. In particular, Shinji et al.
(36) demonstrated that LPS induced the selective
translocation of PKC-ß, Shapira et al. (37) using
isoform-specific Abs identified PCK- as the major isoform involved;
and Herrera-Velit et al. (38) demonstrated that LPS
induced the selective translocation of PKC-.
In this study, we compared the release of TNF- from alveolar
macrophages of young and old rats. The capacity of macrophages from old
rats to secrete TNF- was significantly less than that of macrophages
from young rats, despite the fact that the ability of macrophages to
express the LPS receptor was not impaired in old rats. Owing to the
reputed role of RACK1 in PKC-mediated events, we have examined whether
the lack of TNF- release after LPS stimulation in aged alveolar
macrophages might correlate with changes in RACK1 protein
concentrations. We demonstrated that the age-associated decline in
macrophage functions reflects indeed an impaired PKC signal
transduction pathway and in particular correlates with a defective PKC
anchoring system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The experiments were performed with young (3- to 6-month-old), middle-aged (9- to 12-month-old) and old (>19 month-old) male Sprague-Dawley rats (Charles River, Calco, Italy). All animal care procedures were in accordance with the local Animal Care Committee, and no weight loss or decease was observed after receipt of rats in our animal facility. Rats were housed 2–3 per cage over wood-chip bedding and were allowed food and water ad libitum. Before sacrifice rats were quarantined for 2 wk and were acclimatized to a 12-h light-dark cycle.
Chemicals
LPS from Escherichia coli serotype
0127:B12 was obtained from Sigma (St. Louis, MO). Recombinant murine
TNF- (specific activity, 4 x 107
U/mg) and rabbit anti-murine TNF- were obtained from Genzyme
(Cambridge, MA); Abs against RACK1 and murine CD14 were obtained from
Transduction Laboratories (Affinity, Nottingham, U.K.) and Santa Cruz
Biotechnology (Santa Cruz, CA), respectively; those raised against
PKC- and PKC-ßII have been previously characterized
(39). Salts were purchased from Carlo Erba (Milan, Italy),
and electrophoresis reagents were from Bio-Rad (Richmond, CA). All
reagents were purchased at the highest purity available.
Cells
Alveolar macrophages (AM) were collected by lavaging the lungs as described previously (40). Recovery ranged from 10 to 15 x 106 cells/animal, of which >98% were macrophages as determined by the Giemsa stain. Once washed and resuspended to 106 viable AM/ml, AM were allowed to adhere to plastic plates in serum-free RPMI 1640 (Sigma) containing 2 mM L-glutamine, 0.1 mg/ml streptomycin, 100 IU/ml penicillin, and 50 ng/ml gentamicin (medium). After adherence for 1 h at 37°C in 5% CO2, the plastic plates were washed once with warm medium to remove nonadherent cells. Cells were then exposed to medium with 10% FCS (Sigma) and incubated with or without LPS at the concentration and times indicated.
Oligonucleotide treatment
A 22-base-long antisense oligonucleotide (Primm, Milan, Italy)
corresponding to nucleotides 87–109 in the rat RACK1
sequence (41) was utilized: 5'-TTGCTCGGTCATGGCGTCAGAT-3'
(anti-RACK1). As control, the sense complement oligonucleotide was
used: 5'-ATCTGCAGCCATGACCGAGCAA-3' (senseRACK1). Both
oligonucleotides contained phosphorothioate linkages to limit
degradation. The transfection procedure was adapted from
that of Locati et al. (42). Briefly,
106 AM/ml after adherence were cultured in the
presence or absence of oligonucleotides (5 µM) in medium for 4
h. A final concentration of 10% FCS was then added, and AM were kept
in culture for additional 48 h. Oligonucleotide treatment and
culture conditions were not cytotoxic for the cells as assessed by
lactate dehydrogenase leakage (data not shown). For TNF- release,
after 48 h of incubation the monolayers were washed once with PBS,
and then fresh medium supplemented with 10% FCS and LPS, 10 ng/ml, was
added for 1 h. For Western blot analysis of RACK1, AM were washed
once with PBS and scraped in PBS; after centrifugation, the pellet was
lysed in homogenization buffer as described below.
Assay for TNF
TNF content was assayed by determining the cytotoxicity of TNF against sensitive L929 cells, as previously described (43). The results are expressed in picograms per milliliter or nanograms per milliliter. TNF concentration was calculated against a standard curve with known amounts of recombinant murine TNF.
Western blot analysis
For CD14, PKC isoforms, and RACK1, 10 x
106 AM obtained from rats of different ages were
lysed in 100 µl of homogenization buffer (50 mM Tris, 150 mM NaCl, 5
mM EDTA (pH 7.5), 0.5% Triton X-100, 50 µM PMSF, 2 µg/ml
aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin) and denatured
in 100 µl Laemmli sample buffer (44) for 5 min at
100°C. The protein content of the cell lysate was measured using a
commercial kit (Bio-Rad). For TNF- released in conditioned medium,
4 x 106 AM, after adherence, were cultured
in medium with LPS, 10 ng/ml, for 24 h. The conditioned medium was
quantitatively precipitated by the deoxycholate/trichloroacetate
procedure described by Racchi et al. (45) and resuspended
in 20 µl of Laemmli sample buffer and denatured. The intracellular
proteins (10 µg) were then electrophoresed into a 12%
SDS-polyacrylamide gel under reducing conditions, whereas the
extracellular proteins were electrophoresed into a 18% gel. After
electrophoresis, the proteins were transferred to nitrocellulose
membrane (Amersham, Little Chalfont, U.K.) in 20 mM Tris, 150 mM
glycine, 20% methanol, 0.03% SDS. The different proteins were
visualized with a CD14 antiserum diluted at 1:200, RACK1 at 1:2500,
PKC- at 1:1250, PCK-ßII at 1:5000, and
TNF- at 1:250 as the primary Abs and developed using enhanced
chemiluminescence according to the manufacturer’s instruction
(CDP-Star, NEN, Boston, MA). Molecular weight references were obtained
by running one lane with prestained standard (Amersham).
In vitro PKC-ßII translocation assay
AM (5 x 106) obtained from young and old rats were preincubated in a water bath at 37°C for 30 min in medium with 10% FCS in 15 ml polypropylene tubes to acclimate them. Then, LPS, 100 ng/ml, was added; after 5 and 10 min, AM were recovered by centrifugation for 5 min at 1200 rpm at 4°C. The pellets were resuspended in 500 µl of homogenization buffer (see Western blot analysis) without Triton X-100 utilizing a Teflon/glass potter. Cytosolic fractions were separated by centrifugation at 100,000 x g for 60 min. The pellets were resuspended in the same volume of homogenization buffer by sonication for 2 x 15 s; this constituted the membrane + cytoskeleton fractions.
Reverse transcriptase-polymerase chain reaction
For determination of TNF- mRNA levels, 4 x
106 AM from young or old rats were preincubated
overnight in medium with 10% FCS to avoid adherence induction of
TNF- gene expression. After this time, fresh medium with or without
LPS, 100 ng/ml, was added for 2 h. Total RNA was isolated by
guanidinium thiocyanate-phenol-chloroform extraction (46).
RT-PCR reactions were performed as previously described
(47). Commercially available PCR primers for TNF- and
ß-actin were purchased from Clontech Laboratories (Palo
Alto, CA) and contained the following sequences. Mouse TNF-: sense,
5'-ATGAGCACAGAAAGCATGACCGC-3'; antisense,
5'-CCAAAGTAGACCTGCCCGGACTC-3'. Mouse ß-actin: sense,
5'-GTGGGCCGCTCTAGGCACCAA-3'; antisense,
5'-CTCTTTGATGTCACGCACGATTTC-3'.
The amplified PCR products from cytokine RNA are 692 bp for TNF- and
540 bp for ß-actin. In preliminary experiments, RNA concentrations
and PCR cycles were titrated to establish standard curves to document
linearity and to permit semiquantitative analysis of signal strength (5
and 50 ng for ß-actin and TNF-, respectively). Gels were
photographed with type 55 film (Polaroid, Cambridge, MA). The image of
the PCR products was acquired with a Nikon CCD video camera module
(Nikon, Melville, NY). The optical density of the bands was calculated,
and the peak area of a given band was analyzed by means of the Image
1.47 program for digital image processing (Wayne Rasband, Research
Service Branch, National Institute of Mental Health, National
Institutes of Health, Bethesda, MD).
Statistical analysis
All experiments were performed at least twice; representative results are shown. Statistical significance was determined by Student’s t test or Dunnett’s multiple comparison test as indicated, after ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production from LPS-stimulated AM from old rats
AM from young (3- to 6-month) or old (>18-month) male rats were
obtained by bronchoalveolar lavage. The population recovered, which
consists of >98% macrophages from young and old rats, was tested for
TNF- production in response to increasing concentrations of LPS
(0–100 ng/ml). Fig. 1
shows that AM from
old rats produced at least 50% less TNF- than did AM from young
rats at all concentrations of LPS tested. AM produced on average
12.5 ± 1.5 vs 3.2 ± 0.6 (1 ng/ml of LPS), 17.6 ± 1.0
vs 9.5 ± 4.5 (10 ng/ml of LPS), and 42.8 ± 6.4 vs 13.1
± 1.3 (100 ng/ml of LPS) ng/ml of TNF- from young vs old
rats.
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from old AM was always less
than the release from young AM when stimulated with LPS (Fig. 2).
This diminished capacity of AM from old rats to release TNF-
was confirmed by Western blot analysis of TNF- immunoreactivity in
conditioned medium from AM stimulated with LPS, 10 ng/ml (Fig. 1
, inset). In the absence of LPS, no TNF- release could be
detected by Western blot analysis (data not shown). Furthermore,
the defective TNF- release from old AM was associated with a
decrease in TNF- mRNA expression as assessed by semiquantitative
RT-PCR analysis (Fig. 3), suggesting a
defect in the ability of AM to be activated. The quantification by
densitometric analysis of the ratio of TNF- to ß-actin mRNA
expression indicates a decrease of 40% in TNF- expression in AM
from old rats stimulated with LPS (1.5 ± 0.3 vs 0.6 ± 0.3
of TNF-/ß-actin from young vs old rats).
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than AM from young rats. Comparable amounts of CD14 and PKC isoforms, in the presence of a defective PCK translocation in old rats
We then explored the possibility that this diminished capacity of
AM from old rats to produce TNF- might be due to decreased levels of
LPS receptor (CD14) or from a defect in the activation of AM. To test
the first hypothesis, we compared CD14 expression in cell homogenates
from young, middle-aged, and old rats by Western blot analysis of CD14
immunoreactivity. As shown in Fig. 4 a
comparable or slightly higher CD14 expression was observed in old vs
young AM. No evident difference in CD14 expression was observed in AM
from middle-aged rats.
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and ßII) by Western blot
analysis. As shown in Fig. 4, aging was not associated with changes in
PKC isoform expression. Coomassie blue staining of gel reveals that
equivalent amounts of proteins have been loaded onto gel (Fig. 4). To
further investigate a possible defect in the signal transduction
machinery, experiments of PKC translocation were performed. As shown in
Fig. 5, LPS stimulation induced the
translocation of PC-ßII from the cytosol to the
particulate fraction in young AM, whereas under the same experimental
conditions no translocation of PKC-ßII was
observed in old AM.
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production in old AM was not caused by a
decline in CD14 or total PKC expression, but it was correlated with a
defective PKC-ßII translocation. A deficit in RACK1 contributes to the functional impairment in aged AM
RACKs are emerging as important proteins that bind activated PKCs and anchor them to specific intracellular sites where target substrates can be phosphorylated. As such, RACKs may serve to control PKC-related functions. RACK1 has been reported to bind with high affinity PKC-ßII, which selectively translocated in response to LPS in macrophages.
Fig. 6 inset depicts a representative Western blot of RACK1
immunoreactivity in cell homogenates of AM from rats of different ages.
The Ab recognizes an immunoreactive species at 36 kDa. An
age-related decline in RACK1 expression is evident, which parallels a
decline in the ability of these cells to release TNF- in response to
LPS (Fig. 5). The densitometric analysis showed a decrease of 48
± 2% and 51 ± 1% in RACK1 expression in middle-aged and old
rats vs young rats (p < 0.01).
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production, AM from young rats were treated with RACK1 antisense
oligonucleotide to decrease RACK1 expression, and then TNF- release
was measured after stimulation with LPS. As shown in Fig. 7, inset, under our
experimental condition, a 30% (by densitometric analysis) decrease
in RACK1 expression was obtained, which was associated with a
significant decrease (40%) in TNF- release after 1 h of
treatment: 183 ± 14 vs 312 ± 43 pg/ml TNF- from
antisense vs LPS-treated AM (p < 0.01).
Control sense oligonucleotide did not affect RACK1 expression or
TNF- release (Fig. 7).
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release in AM. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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after LPS
stimulation and to reveal the molecular mechanism(s) underlying
immune senescence. It has been demonstrated (11) that
aged monocytes display a decrease in IL-6 and TNF- secretion,
deficient PKC translocation (, ßI, and ßII), and
mitogen-activated protein kinase activation processes, and decreased
expression of c-Fos and c-Jun. Our results demonstrate, for the first
time, a decrease with age in the PKC-anchoring protein RACK1 expression
in rat alveolar macrophages in spite of no change in levels of
PKC-ßII or PCK-, confirming that
age-associated decline in macrophage functions reflects a defective PKC
signal transduction pathway, specifically affecting the ability of PKC
to translocate to the physiological anchoring sites. Furthermore, we
demonstrated using antisense oligonucleotide in young macrophages that
RACK1 plays indeed an important role in LPS-induced TNF-
release.
We found that aged macrophages, when compared with young macrophages,
produced less TNF-, independently from the concentration of LPS used
or the time considered. This decrease was associated with a decrease in
the mRNA expression of TNF-, as assessed by RT-PCR analysis.
LPS is a potent activator of monocytes/macrophages. The major cell
surface receptor for LPS on monocytes/macrophages is CD14. The
LPS-deficient production of TNF- observed in aged AM is not the
results of a deficiency in the CD14 expression, as demonstrated by a
comparable expression of CD14 in cell homogenates in old vs young
AM.
We then investigated the signal transduction elements of aged cells,
and, in particular, we focused our attention on PKC, which plays an
essential role in LPS-induced TNF- production (27, 28, 29).
We demonstrated that RACK1 expression was deficient in aged alveolar
macrophages in the presence of normal PCK isoform expression. RACK1
immunoreactivity was decreased in old macrophages by roughly 50% when
compared with that in young macrophages. No changes were observed in
the immunoreactivity of PKC- and PKC-ßII,
despite the alteration in RACK1 levels. The RACK1 immunoreactivity and
TNF- release show a progressive decrease with aging. RACK1 is
essential in PCK anchoring to specific intracellular membranes where
target substrates can be phosphorylated. As such, RACKs serve to
control PKC-related functions We speculate that, because of the
decreased RACK1 level, PKC does not bind to the membrane fraction in
aged macrophages on LPS stimulation, resulting in a defective TNF-
production. These speculations are justified by the defective
translocation of PKC-ßII that we observed in
our system and are further supported by the literature, where a
reduction in PKC translocation has been described in different immune
cells (10, 11) and in particular, the data of Delpedro et
al. (11), which demonstrated a defective translocation of
PKC-, PCK-ßI, and
PCK-ßII isoforms in human monocytes.
The decrease observed in RACK1 expression in macrophages or the described deficiencies in PKC activation (10, 11) are probably not limited to the cells of the immune system. Indeed, it has been recently demonstrated a similar alteration in rat brain (48), where a defective PKC signal transduction system in the cortex of aged rats was described. In particular, this deficit was associated with a reduction in the levels of RACK1 anchoring protein and in PKC translocation (49), rather than changes in specific PKC isoform levels (50).
Macrophages are considered surveillance cells of the immune system. They are the first cells to encounter foreign elements (i.e., bacteria, virus, parasites, particles, etc.). Any deficiency in macrophage function will damage the individual. Understanding the age-associated immune function decline is of primary importance better to protect elderly people from infections and cancer. Here, we demonstrated that one of the causes of the deficiency in signal transduction observed in aged macrophages is a decrease in RACK1 expression, essential for PKC functions. At present, however, it is not clear what causes this decrease. A hormonal change in the cell environment might be responsible for the loss of RACK1. To our knowledge, however, the responsive elements in the promoter region of RACK1 have not yet been identified. This will help us better to understand the molecular mechanism(s) involved in immunosenescence and will open the way to exploration of the possibility of repairing age-associated immunological decline.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Emanuela Corsini, Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: PKC, protein kinase C; RACK, receptor for activated C kinase; AM, alveolar macrophages.
Received for publication February 10, 1999. Accepted for publication July 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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