By
§
From the * Laboratory of Biomedical Science, Department of Surgery (Neurosurgery), North Shore
University Hospital, Manhasset, New York; the Department of Emergency Medicine, North Shore
University Hospital, Manhasset, New York; and § The Picower Institute for Medical Research,
Manhasset, New York
The local production of proinflammatory cytokines mediates the host response to inflammation, infection, and injury, whereas an overexpression of these mediators can injure or kill the
host. Recently, we identified a class of multivalent guanylhydrazone compounds that are effective
inhibitors of proinflammatory cytokine synthesis in monocytes/macrophages. The structure of
one such cationic molecule suggested a molecular mimicry with spermine, a ubiquitous endogenous biogenic amine that increases significantly at sites of inflammation and infection. Here,
we addressed the hypothesis that spermine might counterregulate the innate immune response
by downregulating the synthesis of potentially injurious cytokines. When spermine was added
to cultures of human peripheral blood mononuclear cells stimulated with lipopolysaccharide
(LPS), it effectively inhibited the synthesis of the proinflammatory cytokines tumor necrosis
factor (TNF), interleukin-1 (IL-1), IL-6, MIP-1, and MIP-1
. The inhibition of cytokine
synthesis was specific and reversible, with significant inhibition of TNF synthesis occurring even when spermine was added after LPS. The mechanism of spermine-mediated cytokine
suppression was posttranscriptional and independent of polyamine oxidase activity. Local administration of spermine in vivo protected mice against the development of acute footpad inflammation induced by carrageenan. These results identify a distinct molecular counterregulatory role for spermine in downregulating the monocyte proinflammatory cytokine response.
During the early immune response to infection or injury, macrophages synthesize proinflammatory cytokines, which orchestrate the inflammatory reaction. Relatively small amounts of these cytokines produced locally in
tissues benefit the host by activating antimicrobial pathways
and stimulating tissue repair. Evidence of these protective mechanisms has been obtained in animal studies, where administration of anti-TNF antibodies worsens the severity
and duration of Leishmania infection in mice (1), and mice
rendered insensitive to TNF by knockout of TNF receptors are exquisitely sensitive to infection by intracellular
pathogens (2). On the other hand, the uncontrolled release
of larger amounts of cytokines, and the resultant mediator cascade, signals the onset of tissue injury and lethal shock
(3). This potentially disastrous scenario is normally prevented by endogenous counterregulatory mechanisms that
have evolved to inhibit cytokine overproduction. One class
of endogenous cytokine synthesis inhibitors are the glucocorticoid hormones, which are produced during the stress
response, and suppress immune activation and cytokine
synthesis (6, 7). Another class is comprised of the anti-
inflammatory cytokines (e.g., TGF- The present study originated from our recent work focused on a class of low molecular weight multivalent guanylhydrazone compounds that suppress proinflammatory
cytokine synthesis in activated monocytes/macrophages
(15, 16). One of these, N,N Spermine, a ubiquitous biogenic amine that is positively
charged at physiological pH, has been widely studied for its
biological roles in the regulation of DNA synthesis, cellular
proliferation, modulation of ion channel function, and as a
second messenger in cellular signaling (18). A large body of
evidence also implicates spermine as an inhibitor of immune responses. For example, spermine prevents nitric oxide
(NO)1 production in macrophages activated by bacterial endotoxin (19, 20), downregulates human neutrophil locomotion (21), and is immunosuppressive to T cells (22). Increased spermine levels have been measured in tissues
following injury, inflammation, and infection, derived in
part from the release of intracellular spermine from dying
and injured cells, and in part by stimulated biosynthesis
(23). It has been suggested that the accumulation of spermine, and the products of its oxidative metabolism via polyamine oxidase, mediate the anti-inflammatory activity
found in inflammatory exudates, human pregnancy serum,
plasma from arthritic rats, and human rheumatoid synovial
fluid (21, 24). Although these and other studies implicate spermine in suppressing the innate immune response,
it was unclear whether it might also counterregulate proinflammatory cytokine synthesis.
We show here that spermine effectively suppresses the
synthesis of proinflammatory cytokines in human PBMCs.
The mechanism of cytokine inhibition by spermine was post
transcriptional, reversible, specific, and independent of polyamine oxidase activity. The in vivo application of spermine
protected mice against the development of carrageenan-
induced edema, giving evidence that spermine accumulation in tissues can counterregulate the acute inflammatory response.
Cell Isolation and Culture.
Human PBMCs were obtained by
elutriation from normal individual donors to the Long Island Blood
Bank services (Melville, NY). PBMCs were isolated by density
gradient centrifugation through Ficoll (Ficoll-Paque PLUS, Pharmacia, Piscataway, NJ) as previously described (16, 29) with a yield
typically of 2 × 108 adherent cells per isolation. Cells were resuspended in RPMI-1640 (GIBCO BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated human serum, 0.1% L-glutamine, and 0.01% gentamicin, seeded into either 24-well (at 5 × 106 cells/well, for RNA isolation) or 96-well plates (at 5 × 105
cells/well, for spermine uptake and cytokine assays), and cultured overnight at 37°C in a humidified atmosphere of 5% CO2 and
95% air. Nonadherent cells were removed after overnight culture,
and adherent cells (monocytes) were subjected to different experimental treatments as indicated. For studies of murine macrophage-like cells, RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD), seeded
into 24-well tissue culture plates (1 × 106 cells/ml RPMI 1640, 10% FBS, 2 mM glutamine) and subjected to experimental treatments as described for human PBMCs.
Cytokine Induction.
To stimulate cytokine production from
human PBMCs, freshly sonicated Escherichia coli endotoxin (LPS;
Sigma Chem. Co., St. Louis, MO) was added to a final concentration of 100 ng/ml. In some experiments, IFN- Cytokine Assay.
TNF levels in supernatants and cell lysates of
human PBMCs were determined by ELISA using monoclonal
and polyclonal antibodies raised against human TNF (Picower Institute for Medical Research, Manhasset, NY). Serial dilution of
recombinant human TNF (0-10,000 pg/ml) was used in ELISA
to generate standard curves. In brief, 96-well microtiter plates were
coated with 60 µl of supernatants or standard hTNF solution at
4°C for 8-18 h (or at 37°C for 2 h). After washing with buffer
containing 20 mM Tris-HCl (pH = 7.4), 150 mM NaCl, and
0.05% Tween 20, 60 µl of polyclonal TNF antibodies diluted (at
1:200) in buffer containing 10 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 0.2% Tween 20, and 1% goat serum was added and
incubated at room temperature for 2 h. After several washings
to remove the unbound antibodies, the bound antibodies were
then reacted for 30 min with 60 µl (1:2,500 dilution) alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) (Boehringer Mannheim, Indianapolis, IN). After washing, the amount of specifically bound alkaline phosphatase-conjugated antibodies was
determined by assaying for alkaline phosphatase activity with
freshly prepared p-nitrophenylphospate in diethanolamine buffer
(10 mM diethanolamine and 0.5 mM MgCl2, pH = 9.5). A substrate solution was added into each well and incubated for 30 min
at room temperature. The absorption at 405 nm was determined
with a automatic EIA analyzer. MIP-1 Total RNA Isolation and Northern Blotting.
Total RNA was isolated from human PBMCs by using the BRL TRIzol reagent kit
as instructed by the manufacturer (GIBCO BRL, Gaithersburg,
MD), separated on a 1% agarose gel with 2.2 M formaldehyde, and subsequently transferred to Biotrans nylon membrane (ICN, Aurora, Ohio). After UV cross-linking (at 150 mJ), the membrane was prehybridized at 45°C for 2 h in hybridization buffer
containing 5× Denhardt's, 5× SSC, 50 mM NaH2PO4, 0.1%
SDS, 50% formamide and 250 µg/ml salmon sperm DNA, and
subsequently hybridized overnight at 45°C with [32P]dCTP-
labeled TNF or MIP-1 Carrageenan-induced Footpad Inflammation.
Paw edema was induced by injecting 50 µl To study the effect of spermine on TNF
synthesis, human PBMCs purified by elutriation and adherence were stimulated with LPS (100 ng/ml). Whereas LPS
is a potent inducer of TNF synthesis in these cells, pretreatment with spermine (60 min before LPS challenge) effectively suppressed LPS-induced TNF synthesis (Fig. 1). The
50% inhibitory concentration (IC50) of spermine for TNF protein release was 20 ± 15 µM. Maximal TNF suppression was observed with spermine concentrations
We next studied the kinetics of spermine-mediated suppression of TNF synthesis in human PBMCs. In agreement
with previous results showing that TNF is released within 2 h
after stimulation of monocytes with LPS (31), pretreatment
of monocytes with spermine before LPS, or coadministration of spermine simultaneously with LPS, was maximally
effective in suppressing TNF synthesis (Table 1). However,
significant inhibition was also observed, when spermine was
added for up to 2 h after LPS (Table 1). To explore whether spermine-treated monocytes were capable of recovering
the ability to synthesize TNF after spermine removal, we
measured TNF production by human PBMCs that were
incubated with spermine (35 µM) for 1 h, washed with
PBS, and then incubated in fresh media without added
spermine. TNF synthesis by washed PBMCs remained significantly decreased for up to 4 h after spermine removal
from the cellular milieu (4-h washout, TNF = 1580 ± 376 pg/ml; control TNF = 3694 ± 214 pg/ml, P <0.05). By
24 h after removing spermine, TNF synthesis was fully recovered to a level (TNF = 3211 ± 151 pg/ml) that was comparable to PBMCs that had never been exposed to
spermine. Although recovery of TNF synthetic function by
spermine-treated cells indicated that the mechanism of spermine inhibition of TNF synthesis could not be attributed
to cytotoxicity, we also measured LDH release to assess cell
viability. LDH activity was not increased in the media of
spermine treated cells, even when spermine was added in
concentrations exceeding 100-fold the IC50 for TNF suppression ([spermine, 3 mM] LDH = 18 ± 15 U/ml; versus
controls [spermine, 0 mM] LDH = 21 ± 10 U/ml). Separate experiments using trypan blue exclusion and metabolism of MTT also confirmed that spermine concentrations Table 1.
Kinetics of Spermine-mediated Inhibition of TNF
Synthesis from LPS and IFN- and IL-10), which effectively inhibit macrophage activation and proinflammatory cytokine synthesis and prevent the injurious sequelae
of cytokine excess (8). Lastly, prostaglandin E2, which
accumulates at sites of inflammation, can also suppress TNF
synthesis by increasing intracellular cAMP (13, 14). Together, these molecular mechanisms serve to counterregulate or dampen the inflammatory response, and to prevent
the overabundant production of potentially injurious cytokines during the immune response to invasive stimuli.
-bis[3,5-bis [1(amino-iminomethyl)hydrazono]-ethyl]phenyl]-decanediamide tetrahydrochloride (termed CNI-1493) effectively inhibits TNF
translation and suppresses the production of the pro-inflammatory cytokines IL-1, IL-6, MIP-1
, and MIP-1
in human PBMCs (16, 17). Inhibition of proinflammatory cytokine
synthesis by CNI-1493 is specific, because CNI-1493 does not inhibit synthesis of the anti-inflammatory cytokine TGF-
, nor does it prevent the upregulation of MHC class II induced
by IFN-
(16). By suppressing proinflammatory cytokine
production in vivo, CNI-1493 protects mice against the
lethal effects of endotoxin, and prevents the acute inflammatory response in carrageenan-induced footpad edema (15,
16). We considered it plausible that the cytokine inhibitory
activities of this cationic anti-inflammatory molecule might
be attributable to molecular mimicry with an endogenous molecule(s) that normally participates in counterregulating
cytokine production.
(Boehringer
Mannheim, Indianapolis, IN) was also added to a final concentration of 25 U/ml. To evaluate the effect of spermine on production of cytokines from the stimulated human PBMCs, freshly prepared spermine (Sigma) was added to human PBMCs at various
concentrations 1 h before LPS/IFN-
stimulation. After LPS/IFN-
stimulation, supernatants were harvested and assayed for level of
cytokines by ELISA assays. Lactate dehydrogenase (LDH) release,
trypan blue exclusion, and metabolism of MTT were used to
evaluate cytotoxicity.
and MIP-1
levels were
determined by specific sandwich ELISA as previously described
(30), with the exception that coating (goat) and revealing (rabbit)
antibodies specific for human MIP-1
and human MIP-1
were
used. IL-1
, IL-6, and TGF-
levels were determined by commercially obtained ELISA kits according to the instructions of the
manufacturer (Genzyme, Cambridge, MA). Cell lysates were prepared in lysis buffer (50 mM Tris-HCl, 0.5% NP-40, 150 mM
NaCl, 5 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml
pepstatin, 0.02% sodium azide, pH 7.4).
cDNA probes (using Radprime DNA Labeling System from BRL) in the same hybridization buffer. After two washings at room temperature for 20 min with 2× SSC,
0.1% SDS, two washings at room temperature for 20 min with
0.5× SSC, 0.1% SDS, and a final washing at 60°C for 30 min
with 0.1× SSC, 0.1% SDS, the membrane was exposed to X-ray
film at
70°C overnight and developed to visualize the signal.
Level of TNF protein in the supernatants of human PBMCs were
determined by ELISA for comparison to the corresponding TNF
mRNA level.
-carrageenan (0.2% in phosphate buffer
[1× PBS, pH 7.4]; Sigma, St. Louis, MO), into the plantar surface of the left hind footpad of female C3H/HeN mice (20-25 g
body weight) either alone, or in combination with various concentrations of spermine. The right hind footpad was injected with
50 µl of PBS as control. 28 h after footpad injection, the thickness of the carrageenan- and saline-injected footpad was measured
using calipers, and the data expressed as the difference between
the diameters of the two footpads.
Spermine Suppression of TNF Synthesis in LPS-activated
Human PBMCs.
100 µM,
which suppressed TNF production to levels that were 25-
35% of the TNF levels produced by controls. Spermine did
not interfere with ELISA detection of TNF as evidenced by the fact that TNF standard curves measured in the presence of spermine were comparable to TNF standard curves
measured in its absence (data not shown). Moreover, TNF
levels as assessed by the L929 cell cytotoxicity bioassay were
also inhibited by addition of spermine (data not shown).
Although the purified human PBMCs contained more than
95% monocytes, we wished to confirm that spermine inhibited TNF production in the murine macrophage-like cell line RAW 264.7. In these experiments, we observed
that spermine inhibited LPS-induced TNF synthesis in
RAW cells, and that the IC50 was 40 ± 20 µM, a value comparable (P >0.05) to the IC50 determined in human PBMCs
(see above).
Fig. 1.
Spermine inhibits TNF synthesis from LPS-stimulated human PBMCs. Human PBMCs were exposed to the concentrations of
spermine as indicated for 1 h, then stimulated by addition of LPS (100 ng/ml) and IFN-. TNF released into the conditioned supernatants collected after 4 h was measured by ELISA and results expressed as percentage of control (no spermine). Data shown are mean ± SEM of three independent experiments. (Control TNF = 7416 ± 763 pg/ml.)
[View Larger Version of this Image (10K GIF file)]
3 mM were not toxic to human PBMCs during a 4 h
treatment (data not shown).
-stimulated Human PBMCs
Time of spermine addition (min)*
TNF (percent of controls)
No spermine added
100
60
26 ± 2
30
26 ± 4
5
33 ± 3
0
28 ± 3
+5
21 ± 3
+30
52 ± 7
+60
49 ± 8
+120
66 ± 7
*
LPS and IFN- were added at time 0. Spermine (35 µM) was added
at the relative times shown, and TNF in supernatants conditioned for 4 h after LPS was detected by ELISA. Data are mean ± SEM from six
replicates of an experiment that was repeated twice.
The mechanism of spermine action on cytokine
synthesis was initially investigated by Northern blot analysis
of total RNA extracted from LPS-activated human PBMCs
using [32P]dCTP-labeled TNF and MIP-1 cDNA as
probes. In agreement with previous results (17), TNF and
MIP-1
mRNA were not detected in human PBMCs in
the absence of LPS, but were significantly increased 2 h after addition of LPS (100 ng/ml) (Fig. 2). Steady state levels of TNF and MIP-1
mRNA were not decreased by addition of spermine at a concentration (35 µM), although
TNF and MIP-1
protein synthesis in the same cell cultures was suppressed to a level that was just 25 and 8% of
controls, respectively (P <0.05) (Fig. 3, a and c). We obtained similar results in separate experiments using quantitative PCR methodology in which the steady state level of
TNF mRNA was found to be comparable between spermine-treated (35 µM) and untreated LPS-stimulated PBMCs,
whereas spermine inhibited TNF protein synthesis in the
same cells (data not shown).
To define further the posttranscriptional action of spermine on the release of TNF and MIP-1, we assayed the
cellular levels of these cytokines in lysates of LPS-stimulated cells. Exposure of human PBMCs to spermine significantly inhibited the levels of cell-associated TNF and MIP-1
(Fig. 3, b and d). We observed a similar IC50 for spermine
inhibition of cell-associated and secreted cytokine levels.
Because the levels of cell-associated cytokines were diminished and not increased, these results provide direct evidence
that the mechanism of spermine action is by inhibiting cytokine synthesis, and not through suppressing cytokine release.
The specificity of spermine inhibition of proinflammatory cytokine synthesis was determined by measuring the
levels of other macrophage-derived cytokines in the supernatants of LPS-activated human PBMCs. We observed spermine dose-dependent suppression of the proinflammatory
cytokines MIP-1, IL-1
, and IL-6, from LPS-activated human PBMCs (Fig. 4). The IC50 for spermine inhibition
for each of these four proinflammatory cytokines was ~2
µM, with maximal suppression of MIP-1
, MIP-1
, and
IL-6 exceeding 90%. However, the effects of spermine on
IL-1
synthesis differed somewhat from these other cytokines, because at saturating spermine concentrations, IL-1
synthesis was only decreased by 65% as compared with
controls. The residual amount of IL-1
synthesis (35%) that
could not be inhibited by spermine treatment was similar
to the residual amount of TNF synthesis that persisted in
the presence of spermine (see above). Additional evidence
for the specificity of spermine inhibition of proinflammatory cytokine synthesis was given by the observation that
spermine failed to suppress the constitutive production of TGF-
from human PBMCs (Fig. 4 d), even when spermine was added in concentrations more than 100-fold
higher than the IC50 for the proinflammatory cytokines.
When considered together, these results indicate that spermine specifically and reversibly inhibits proinflammatory
cytokine synthesis in human PBMCs.
Spermine Inhibits TNF Synthesis in Serum-free Media and in the Presence of Polyamine Oxidase Inhibition.
Szabo and coworkers (19, 20, 32) recently reported that spermine suppressed the induction of inducible nitric oxide synthase (iNOS) in the cell line J774.2, and found that the molecular basis for this suppression was dependent upon oxidation of spermine by polyamine oxidase present in bovine serum. To address the possible role of polyamine oxidase-mediated spermine oxidation in the mechanism of spermine action on inhibiting proinflammatory cytokine synthesis in primary human monocytes, we measured TNF production in human PBMCs that had been cultured and stimulated with LPS under serum-free conditions. We observed a spermine dose-dependent suppression of TNF in the absence of added serum (Fig. 5 a), and determined that the IC50 under serum-free conditions was similar to that measured in serum-containing media (serum-free IC50 = 8 ± 4 µM) versus serum-containing media (10% FBS IC50 = 20 ± 15 µM; P >0.05). Although these experiments suggested that oxidation of spermine by polyamine oxidase was not required for TNF suppression in human PBMCs, we wished to confirm this interpretation by adding a pharmacological inhibitor of polyamine oxidase to serum-containing media and then measuring the effect of spermine on TNF synthesis. In these experiments, we measured spermine oxidase activity in bovine serum-containing media using HPLC detection of spermine, and in agreement with previous results (33), addition of aminoguanidine (2 mM) suppressed enzyme activity to <1% of controls. In these conditions of complete enzyme inhibition we also observed a spermine dose-dependent suppression of TNF synthesis (Fig. 5 b). The IC50 for spermine inhibition of TNF in the presence of complete polyamine oxidase inhibition with aminoguanidine (IC50 = 8 ± 6 µM) was similar to that observed in controls, as measured in serum-containing media without addition of aminoguanidine (IC50 = 20 ± 15; P >0.05). Although these results suggested that spermine oxidation is not required for the cytokine suppressing activity of spermine, we wished to control for the possibility that an alternative oxidative mechanism might have resulted in the formation of spermidine in these cultures. Accordingly, we assessed the effects of spermidine on LPS-stimulated TNF synthesis. Although we did observe spermidine to inhibit TNF synthesis in human PBMCs, the measured IC50 (638 ± 143 µM) was significantly higher than for spermine itself (P >0.05), suggesting that metabolism of spermine to spermidine is not the molecular basis for the observed cytokine inhibition by spermine. When considered together, these results indicate that the inhibition of proinflammatory cytokine synthesis in human PBMCs is directly attributable to spermine, and is not dependent upon the activity of polyamine oxidase.
Administration of Spermine In Vivo Suppresses Carrageenanmediated Inflammation.
The observations that spermine decreases proinflammatory cytokine production by LPS-stimulated human monocytes suggested that local accumulation
of spermine at inflammatory sites could suppress the development of cytokine-mediated tissue responses. We investigated this mechanism in vivo by measuring the development of edema induced by injection of carrageenan into
the footpad of mice, a widely used model of inflammation
in which cytokine antagonists and other anti-inflammatory
agents can effectively suppress the inflammatory response
(15, 34, 35). Administration of spermine directly into the
carrageenan-injected paw significantly suppressed the development of swelling (Fig. 6). The anti-inflammatory effects were spermine dose-dependent, and maximally inhibited
footpad swelling by 48% as compared with vehicle-treated
controls (P <0.05). Because the development of acute
edema in this model is dependent upon the activity of cytokines (and independent of LPS), these results now suggest
that the local accumulation of spermine in tissues can suppress the development of injurious inflammation in vivo.
Some 44 years ago, a search for a natural product in animal tissues capable of suppressing the growth of tubercle bacilli ultimately led Hirsch and Dubos to discover that spermine was the active anti-mycobacterial principle (36). This seminal work revealed a potential mechanism through which the cytotoxic activity of a ubiquitous polyamine could protect the host during invasive infection. Because it was already known that spermine concentrations were significantly elevated in tissues during infectious, neoplastic, and inflammatory diseases (e.g., tuberculosis, pneumonia, cancer), they proposed a direct role for spermine in limiting the growth or spread of an infectious agent or tumor (36). Our present results now support an additional mechanism by which spermine can participate in the host response to infection or invasion, by counterregulating proinflammatory cytokine synthesis.
The proinflammatory cytokines TNF, IL-1, IL-6, MIP1, and MIP-1
occupy a pivotal role in stimulating the
early stages of acute inflammation, including recruitment and
activation of inflammatory cells, stimulation of endothelial
cell activation, and direct cytotoxicity (5, 37). Whereas
these inflammatory events can be critical to the ultimate recovery from infection or injury, normal counterregulatory
mechanisms are also critical to the success of the immune
response, because the inappropriate or excessive production of proinflammatory cytokines ultimately can lead to the development of shock and tissue injury (3, 4, 40). Previously, extensive investigations of counterregulatory immune mechanisms have focused on the cytokine inhibitory
roles of the glucocorticoid hormones, the anti-inflammatory cytokines TGF-
and IL-10, and the prostaglandin
PGE2 (8, 14, 41). Together, these studies demonstrate the critical central role occupied by counterregulating
signals that serve to dampen the immune response. The
present results now indicate that spermine counterregulation of proinflammatory cytokine production offers another level of molecular regulation capable of reducing the
injurious activity of a local immune response.
In agreement with its proposed cytokine counterregulatory role, spermine-mediated cytokine inhibition was specific, reversible, and time dependent, therefore enabling an
innate mechanism in which the affected monocytes can recover their cytokine producing function to participate in
subsequent immune responses. Because spermine effectively inhibited cytokine synthesis in serum-free conditions,
and in the presence of the polyamine oxidase inhibitor aminoguanidine, oxidative metabolism of spermine is not
required for the molecular mechanism of cytokine counterregulation. Spermine levels required to suppress cytokine synthesis are readily achieved in vivo, because high
tissue concentrations (500 µM to 2 mM) have been reported in tumors, and in patients infected with bacteria, mycobacteria, and viruses (36, 44). Because elevated
polyamine levels have also been implicated in the immunosuppressive state associated with pregnancy and fetal development (24, 48, 49), it is now interesting to consider
whether the mechanism of immunosuppression in these
earlier observations is attributable at least in part to spermine-mediated counterregulation of proinflammatory cytokines. For instance, elevated spermine levels found in
amniotic fluid might dampen the production of TNF and
IL-1, and thereby prevent the onset of abortion mediated
by cytokine release in the amnion (50). The present study
did not address the role of spermine on T cell cytokine
synthesis, but earlier work by Flescher and colleagues (51)
suggested that spermine does not inhibit IL-2 production
in PHA-stimulated monocytes. Rather, inhibition of IL-2
synthesis was observed by a combination of spermidine,
polyamine oxidase, and hydrogen peroxide, leading them
to conclude that oxidative metabolism of spermidine, but
not spermine, can inhibit T cell IL-2 production (51). In
contrast, we found that spermidine was a poor inhibitor of
monocyte/macrophage proinflammatory cytokine synthesis, and that spermine was an effective inhibitor even in the
absence of polyamine oxidase activity. When considered together, these findings now suggest that spermine suppression of monocyte proinflammatory cytokine synthesis is specific and direct.
It will be of interest to investigate whether spermine
inhibition of cytokine translation is dependent upon cellular signaling pathways that are shared with other cytokine
counterregulators, and whether they converge on some key
regulatory step(s). We have considered the hypothesis that
spermine might act via a molecular pathway shared with
the glucocorticoids, but the following observations suggest
that spermine-mediated cytokine counterregulation is independent of glucocorticoid signaling. (a) Spermine inhibition
of TNF and MIP-1 occurs posttranscriptionally, whereas
glucocorticoids inhibit both transcription and translation
(7); (b) spermine retains its counterregulatory activity in the
presence of IFN-
, but IFN-
overrides the suppressive activity of glucocorticoids (52); and (c) the recovery of monocyte TNF synthesis in spermine washout experiments is more
rapid than that predicted for glucocorticoid-mediated inhibition (53). These observations suggest that sperminemediated cytokine counterregulation occurs by a molecular
pathway that is distinct from glucocorticoids. It will also be
of interest to address whether the inhibitory effects of spermine in monocytes/macrophages are dependent upon an as
yet unidentified monocyte/macrophage receptor or binding protein at the cell surface, and whether spermine transport into the monocyte/macrophage is required for interaction with an intracellular target.
Previous studies indicate that LPS stimulation of monocytes activates spermine uptake via a protein kinase C (PKC)-
dependent mechanism (15, 54). In agreement with these
results, we have observed enhanced spermine uptake after
LPS stimulation in human PBMCs (data not shown). It is
plausible that LPS-stimulated spermine uptake can participate in the observed suppression of proinflammatory cytokine
synthesis. It should be noted that the action of spermine in
suppressing cytokine synthesis is not simply attributable to neutralizing LPS via a charge effect, because LPS-stimulated signaling in the presence of spermine still produced
significant increases in the levels of TNF and MIP-1
mRNA (Fig. 2). Moreover, spermine effectively suppressed
the development of edema induced by carrageenan in vivo,
an acute inflammatory response that is dependent upon cytokine production but independent of LPS (15, 34, 35).
Spermine counterregulation of proinflammatory cytokine synthesis was specific, because the suppression of MIP1, MIP-1
, and IL-6 approached 100%, but maximal suppression of TNF and IL-1
failed to exceed 75%. In further
agreement with its proposed role as an anti-inflammatory
mediator, spermine failed to suppress TGF-
, a potent antiinflammatory cytokine (8). Consideration of these results leads
to the appreciation that TNF and IL-1
each occupy pivotal
beneficial roles in the immune response, so that the proposed mechanism of spermine in differentially counterregulating cytokine release also enables the continued production of beneficial, or perhaps necessary, low levels of these
two mediators. Even in the presence of the very high spermine levels found in diseased tissues, low level production
of TNF and IL-1 could persist to benefit the host by stimulating antimicrobial and antiviral immune responses,
promoting tissue regeneration, and facilitating wound healing (5). Meanwhile, the uninhibited release of TGF-
can participate in further counterregulation and containment of
the local immune response.
We conclude that spermine counterregulation of cytokine production is a mechanism for locally suppressing the acute, and potentially injurious tissue response to inflammation in vivo. Because spermine is released from dying and injured cells during infection, injury, ischemia, and inflammation, it is suitably positioned as a feedback signal to suppress further damage from cytokine excess. Tissue spermine levels predictably reflect the extent of surrounding tissue injury, thereby providing a sensitive counterregulatory signal to prevent excessive activation of macrophages. This molecular counterregulatory feedback loop would be anticipated to predominate locally at tissue sites of inflammation, and not systemically as is the case with the glucocorticoids and the anti-inflammatory cytokines.
Address correspondence to Kevin J. Tracey, North Shore University Hospital, The Picower Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030.
Received for publication 16 December 1996 and in revised form 28 February 1997.
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