CELL STRUCTURE AND FUNCTION 29: 27–34 (2004)
© 2004 by Japan Society for Cell Biology
Morphological and Biochemical Characterization of Macrophages Activated by
Carrageenan and Lipopolysaccharide In Vivo
Valéria Pereira Nacife1∗, Maria de Nazaré Correia Soeiro2, Rachel Novaes Gomes3, Heloísa D’Avila3,
Hugo Caire Castro-Faria Neto3, and Maria de Nazareth Leal Meirelles1
1Laboratório de Ultra-estrutura Celular, 2Laboratório de Biologia Celular, Departamento de Ultra-estrutura e
Biologia Celular, and 3Lab. de Imunofarmacologia, Departamento de Fisiologia e Farmacodinâmica, Instituto
Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brasil
ABSTRACT. Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus
restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface
charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron micros-
copy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the
light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation
for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular
vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well
as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable
increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and
stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stim-
ulated by LPS produce almost the same quantities of TNF-α, while in vivo stimulation by CAR leads to a 30–
40% increase of cytokine release in vitro compared to the other groups.
In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces
a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory
Macrophages can be divided into normal and inflammatory
macrophages. The former includes macrophages in connec-
tive tissue (histiocytes), liver (Kupffer cells), lung (alveolar
macrophages), lymph nodes, spleen, bone marrow, skin
(histiocytes, Langerhans cells) and others. Inflammatory
macrophages are characterized by their various specific
markers and share similar properties to the monocytes
(Rosenberger and Finlay, 2003; Maurer et al., 2002).
Macrophages play a fundamental role in both humoral
and cellular immune responses. They are able to rapidly rec-
ognize, internalize and destroy a large number of pathogens,
thus restricting the infection until the cell host is able to
initiate adaptive immunity (reviewed in Rosenberger and
Finlay, 2003). They present antigens to lymphocytes, modu-
late T cell functions, and secrete a large number of inflam-
matory mediators, which play roles in the amplification
of both humoral and cell-mediated immune responses.
(Jedynak and Siemiatkowski, 2002).
Due to their varied roles and the environmental stimuli
that they receive, macrophages exhibit different phenotypes,
which are mostly related to their morphology, cell surface
antigen expression and function. This phenotypic hetero-
geneity is a consequence of a series of down-regulations of
certain cellular processes and the up-regulation of others.
(Arandjelovic et al., 1998). The appearance and activation
of macrophages are thought to be rapid events in the devel-
opment of many pathological lesions, including malignant
*To whom correspondence should be addressed: Valéria Pereira Nacife,
Laboratório de Ultra-estrutura Celular, Departamento de Ultra-estrutura e
Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av.
Brasil 4365, 21045-900, Rio de Janeiro, Brazil.
Tel: +55–21–25984641, Fax: +55–21–22604434
Abbreviations: LPS, Lipopolysaccharide; CAR, Carrageenan; CF, Cation-
V.P. Nacife et al.
tumors, atherosclerotic plaques, and arthritic joints.
Recently, macrophages have been used as novel cellular
vehicles for gene therapy (Burger and Dayer, 2002; Greaves
and Gordon, 2002).
In the literature, several agents have been used to pro-
mote macrophage activation, including the lipopolysaccha-
ride (LPS) and the carrageenan (CAR) molecules (Risco
and Pinto da Silva, 1995; Nacife et al., 2000; Tsuji et al.,
2003). LPS is a bacterial endotoxin, which acts on numer-
ous cellular functions through the processes of cell activa-
tion and damage. The molecular mechanisms involved in
the “endotoxic phenomenon” are still not completely under-
stood (Risco and Pinto da Silva, 1995).
Carrageenan is known to induce acute inflammatory exu-
dation (Winter et al., 1962) and paw edema (Vinegar et al.,
1969; Henriques et al., 1987), injury to blood vessels (Ward
and Cochrane, 1965), inhibition of complement functions
(Davies, 1965) and kinin release (Di Rosa and Sorrentino,
1970). It is also used to induce the formation of air pouch
in rodents (Sedgwick and Lees, 1986), followed by cell
accumulation and mediator release (Romano et al., 1997).
Up to now, few data are available concerning the mor-
phological and biochemical aspects related to the cells acti-
vated by CAR, hence our present aim is to characterize
some properties of mice macrophages stimulated by CAR in
vivo through light and electron microscopy assays. Besides,
we also analyzed the release of some inflammatory media-
tors in vitro comparing to the data obtained from another
well-known inflammatory stimuli, LPS.
Material and Methods
Carrageenan λ type IV and LPS (from Escherichia coli) were pur-
chased from Sigma Chemical Co. USA.
In vivo stimulation
Swiss male mice (18–20 g) were intraperitoneally injected with
300 µg CAR or 250 ng LPS in saline as described (Nacife et
al., 2000). In the control group, mice did not receive any type of
Peritoneal cell cultures
Peritoneal cells from the different groups of mice were collected
using Dulbecco’s modified Eagle medium (DME) without serum.
For light microscopy analysis and inflammatory mediators mea-
surements, after the determination of the cell density by Newbauer
chamber quantification, the obtained cells were plated into 24-well
microplates at a cell density of 106 cells/well, with 10% of fetal
calf serum (DMES) or with supplementation of 2% serum bovine
albumin for light and inflammatory mediators measurements,
After 24 and 48 hours of cells plating, untreated and CAR- and
LPS-treated peritoneal cells, were fixed in Bouin’s solution,
stained with May-Grünwald-Giemsa and analysed at light micros-
Transmission electron microscopy (TEM)
Peritoneal cells from untreated mice and after stimulation for 24
and 48 hours with CAR and LPS were collected, fixed with 2.5%
glutaraldehyde in 0.1 M sodium cacodylate buffer and 3.5%
sucrose for 1 hour at 4°C, post-fixed with 1% osmium tetroxide in
the same buffer for another hour at 4°C. They were dehydrated in a
graded series of acetone and embedded in Epon resin. Ultrathin
sections were stained with uranyl acetate and lead citrate and
observed with Zeiss electron microscope (EM 10C).
Surface charge analysis
Peritoneal macrophages from untreated mice and after 24–48
hours of in vivo stimulation with CAR and LPS were collected
using Dulbecco’s modified Eagle medium. The cells were then
washed in cold saline and immediately treated for 30 minutes at
4°C with 100 µg·ml–1 cationized ferritin (CF) and processed for
TEM as described above.
Inflammatory mediators measurements
After 24 and 48 hours of in vivo stimulation by CAR and LPS, the
peritoneal cells were collected as described and seeded in 24-well
microplates at a cell density of 106 cells/well. After 24 and 48
hours of culture plating (grown in the absence of serum sources but
supplemented with 1% albumin serum bovine), the supernatants
were collected and stored at –70°C for analysis of nitric oxide
(NO), tumoral necrosis alpha factor (TNF-α) and prostaglandins
E2 (PGE2) levels, and fresh medium was added to the microplates.
The quantification of NO was done by mixing 50 µl of the culture
supernatant with an equal volume of the Griess reagent in a 96-
well flat-bottomed microtiter plate. This reagent was prepared by
mixing equal volumes of 1% sulphanilamide in water containing
5% phosphoric acid with 0.1% N-(1-naphtyl) ethylenediamine
hydrochloride (Ding et al., 1988). After 15 min at room tempera-
ture, the plates were read at 540 nm. In each individual plate,
duplicates of a sodium nitrite solution in eight concentrations from
0 to 100 µM were used to generate standard curves. For TNF-α
and PGE2 analysis we performed an ELISA assay according to
the manufacturer’s instructions (Duo Set Kit, R&D, Cayman
Chemical, Ann Arbor, MI).
At light microscopy level, cells obtained from the perito-
neum of CAR-injected mice showed a higher degree of
spreading, increased number of membrane projections and a
large number of vacuole profiles (Fig. 1B) as compared to
both untreated (Fig. 1A) and LPS-treated (Fig. 1C) mice.
Macrophage Activation by CAR and LPS
The analysis by TEM of the CAR and LPS-stimulated
macrophages showed similar results after 24–48 hours of
the in vivo treatment and confirmed the light microscopy
results. The peritoneal cells collected after CAR stimulation
displayed a higher number of membrane projections and
cytoplasmic vacuoles (Fig. 2B and 2C) as compared to the
non-stimulated macrophages (Fig. 2A). However, the analy-
sis of the LPS-stimulated cells showed a greater number of
cytoplasmic projections (Fig. 2D) as compared to the con-
trol cells (Fig.2A) but a smaller number of vacuoles as
compared to the CAR-treated mice group (Fig. 2B and 2C).
Since the surface properties of the membranes from the
CAR activated-macrophages are not well understood and
may be involved in the altered morphology observed both
by optical and electron microscopy analysis, we next inves-
tigated their surface charge by means of cationized ferritin
(CF) binding. After incubation for 30 min at 4°C in the pres-
ence of CF, the untreated macrophages were intensively
labeled all over their surface (Fig. 3A). However, stimula-
tion by CAR in vivo resulted in a drastic decrease in the
binding capacity of the cell membrane to the cationized
ferritin particles suggesting a modulatory effect on the
surface charge of macrophage plasma membrane by CAR
(Fig. 3B). On the contrary, the samples collected after LPS
stimulation did not present significant differences (Fig. 3C)
concerning the amount and distribution of the positive
tracer towards the anionic surface sites as compared to the
untreated control (Fig. 3A).
Next, we investigated the release of certain inflammatory
mediators at the supernatant of the macrophages collected
after induction or not by CAR and LPS (Table I). Our
results show that peritoneal cells obtained from non-stimu-
lated mice released increasing amounts of nitrite along with
the length of time of their in vitro culture: about 13.7 and
29.9 µM, after 24 and 48 hours of plating, respectively.
Stimulated-CAR macrophages displayed higher levels as
compared to the control cells reaching 33.2 and 52.6 µM
and displaying rises of 142 and 75% after 24 and 48 h plat-
ing, respectively. On the other hand, LPS group displayed
less increase as compared to CAR treated mice reaching
about 22.1 and 39.4 µM of nitrite release in the culture
supernatant, displaying 61 and 31% of increase as compared
to untreated samples (Table I).
The measurement of PGE2 release in the supernatant
from non-stimulated and stimulated macrophages by CAR
and LPS after 24 hours of culture plating showed levels of
8.8, 15.2 and 9.6 ng/ml, respectively; which proportionally
increased slightly in all groups after 48 hours of plating,
Light microscopy of mice peritoneal cells in Giemsa-stained
slides showing the characteristic morphology of non-stimulated (A), CAR-
stimulated (B) and LPS-stimulated macrophages for 48 hours in vivo. Note
the increase of spreading and cytoplasmatic vacuole profiles (asterisk) in
the CAR-stimulated macrophages (B) as compared to the non-stimulated
(A) and LPS treated mice (C). Bar=25 µm
V.P. Nacife et al.
reaching 10.5, 17.0 and 12.8 ng/ml (Table I). As noted with
nitrite production, the CAR stimulated group displayed
higher levels of PGE2 release as compared to untreated and
LPS treated mice, reaching 72 and 61% of mediator release
after 24 and 48 hours of plating as compared to the cells
obtained from non-stimulated mice. On the other, compar-
ing to non-stimulated group, LPS treatment led to a small
rise of PGE2 release in the supernatant, displaying increases
of 9 and 22% after 24 and 48 hours of cell culture, respec-
The measurement of another inflammatory mediator,
TNF-α, showed that only the CAR stimulated group pre-
sented a small increase in cytokine release (29% and 43% of
increase) in the culture supernatant as compared to the non-
stimulated macrophages, after 24 and 48 hours of plating,
respectively. LPS always showed similar values as com-
pared to cells collected from non-stimulated mice (Table I).
Macrophages function as both sentinels and the first line of
defense against infection, and therefore are an essential bar-
rier that pathogens must overcome to be successful. They
have a fundamental role in humoral and cellular immune
responses being involved in a variety of immunological and
inflammatory processes from antigen presentation to micro-
bicidal and tumoricidal activities (reviewed in Gordon,
1998; Hubbard, 1999).
Our present results showed that in vivo administration of
CAR represents a potent inflammatory stimulation for peri-
toneal macrophages leading to a higher degree of spreading,
an increase in their size, increase in the number of mem-
brane projections and an overall enhancement in the number
and size of intracellular vacuoles as compared to the cells
obtained from untreated and even submitted to another
inflammatory stimuli such as LPS. The ultrastuctural analy-
Ultrastructural aspects of mice peritoneal cells showing the characteristic morphology of non-stimulated (A) LPS-stimulated (D) and CAR-stimu-
lated macrophages for 24 (B) and 48 hours in vivo (C). Note the huge increase in the number of intracellular vacuoles (asterisk) and membrane projections
(arrow) in the CAR-stimulated macrophages (B and C) as compared to the non-stimulated (A) and LPS treated mice (D). Bar=1 µm
Macrophage Activation by CAR and LPS
sis confirmed the light microscopy data and clearly showed
that although these cells displayed altered morphology
mostly due to the presence of large vacuoles and membrane
projections, they were not damaged. They presented plasma
membrane integrity and well-preserved mitochondria pro-
files characteristic of good cell viability. Our results showed
that peritoneal cells collected from the LPS-treated group
also displayed altered morphology, however, it was less
intense as compared to the CAR stimulation. Our present
data confirmed previous report, which showed that CAR
injection did not affect cell viability measured by the incor-
poration of propidium iodide (Nacife et al., 2000). The
authors showed that the increased number of vacuoles
during CAR stimulation was probably due to the increased
exocytosis of inflammatory proteins, which could be
detected in their culture supernatant. In fact, our present
results concerning the measurements of the inflammatory
mediators also showed that CAR induced a higher degree of
inflammatory release as compared to that of mice injected
Macrophages like others vertebrate cells have a negative
charge due to the membrane anionic components (Weiss,
1969; Burry and Wood, 1979; Meirelles et al., 1984;
Mutsaers and Papadimitriou, 1988). The surface charge
plays an important role in cell-cell interactions, cellular
differentiation, adhesion and endocytosis (van Oss, 1978;
Papadimitriou, 1982; Spangenberg and Crawford, 1987; de
Carvalho and de Souza, 1990). In the present work, we
observed that macrophages collected from mice stimulated
by CAR showed a decrease in the number of CF particles in
their surface as compared to the macrophages obtained from
non-stimulated and LPS-stimulated mice. Alterations of the
surface charge also have been described in erythrocytes,
which paralleled changes in their morphology (Samoilov et
al., 2002). On the other hand, macrophages elicited by
thioglycolate did not display any differences concerning the
binding of cationized ferritin particles at pH 7.2 to the cell
surface as compared to resident and macrophages (Santos
and De Souza, 1983). Since the macrophages have an
important endocytic and exocytic activities, which involves
the cell surface properties (Silva Filho and De Souza, 1988;
Mutsaers and Papadimitriou, 1988; Rabinowitz and Gor-
don, 1989), the reduced anionogenicity presently observed
in the CAR-activated macrophages could lead to alterations
in the endocytic/exocytic pathways of these cells, which has
been previously suggested during internalization assays
employing fluorescent probes such as lysotracker yellow
Electron micrograph of macrophages after incubation with cation-
ized ferritin. A remarkable reduction in the number of cationized ferritin
particles (arrow) associated to the surface of CAR-stimulated macrophages
(B) as compared to untreated samples (A) is evident. Conversely, no great
differences were observed in the amount and distribution of the positive
tracer towards the anionic surface sites (arrow) in the LPS-treated group
(C), as compared to the untreated control (A). N=Nucleus. Bar=0.5 µm
V.P. Nacife et al.
and acridine orange (Nacife et al., 2000). Further studies are
now under way to further investigate these pathways in
CAR stimulated cells using specific ligands for the receptor
mediated- and fluid phase endocytosis.
At the site of an inflammatory reaction, injured vascular
endothelial cells and emigrated leukocytes release several
inflammatory mediators, which modulate and maintain
the inflammation. Carrageenan is used as a component to
induce non-specific inflammation, possibly through activa-
tion of innate immune responses. It modulates acquired
immunity, adjuvant and suppressive effects (Coste et al.,
1989; Macino and Morelli, 1983; Bash and Vago, 1980;
Nicklin and Miller, 1984; Cochran and Baxter, 1984; Vijaya-
kumar et al., 1990). In our results, we observed that CAR
stimulated-macrophages displayed a remarkable increase
(142–75%) of nitric oxide production as compared to non-
stimulated cells and a significant increase of about 50–33%
as compared to the macrophages collected from mice stimu-
lated with LPS. The role of NO in the inflammation has
been deeply investigated. NO is generated from L-arginine
by a family of enzymes called NO synthases (NOS) (Morris
and Billiar, 1994). NO has been shown to have pro- and
anti-inflammatory actions (Darley-Usmar et al., 1995). It
has been reported that when NO is generated by the consti-
tutive isoform of NOS in the vascular endothelium, this
messenger inhibits both neutrophil adhesion to the endothe-
lium (McCall et al., 1988) and to the postcapillary venules
(Kubes et al., 1991; Arndt et al., 1993). Regarding carrag-
eenan, it is known that NO increases plasma extravasion,
paw edema (Ialenti et al., 1993) and granuloma formation
(Iuvone et al., 1994) induced by CAR stimuli. In another
study, it has been reported that the intraplantar injection of
this agent increases the levels of NO in the model of paw
edema, which can be inhibited by NO inhibitors, such as
NG-monomethyl-L-arginine and NG-nitro-L-arginine
methyl ester (Salvemini et al., 1996).
Upon stimulation by interferon-γ (IFN-γ), tumor necrosis
factor α (TNF-α) and lipopolysaccharide (LPS), macroph-
ages express a Ca+2/calmodulin-independent NOS, leading
to NO production (Nathan, 1992; Jorens et al., 1995). The
NO produced by macrophages mediates the cytostatic and
cytotoxic effects to a variety of pathogens including viruses,
bacteria, protozoa and helminthes (Sternberg and McGuig-
nan, 1992; Mabbott et al., 1994; Visser et al., 1995; revised
by James, 1995). In our present study, we showed that the
effects of CAR on peritoneal cells reflect its high ability as
an inflammatory stimulus, being superior to the effects of
Besides the analysis of NO release, we also assayed the
PGE2 production by peritoneal cells collected from CAR-
stimulated mice. Prostaglandins are well-known inflamma-
tory mediators (Hunter, 2002; Kast, 2001, Kuraishi and
Ushikubi, 2001) and the literature data showed that mac-
rophages stimulated by CAR can produce PGE2, which
could be responsible for the immunosuppressive activities
of CAR, which is related to the dosage, route of administra-
tion and fraction used to perform the experiments (Bash and
Vago, 1980). In our present analysis we found a striking
increase of PGE2 release in the supernatant of cultured
peritoneal cells after in vivo stimulation as compared to
non-stimulated and LPS-treated mice (70 and 58% of
In inflammatory reactions induced by λ-CAR, the role
of TNF-α has already been investigated. In models of
carrageenan-induced pleurisy it has been noted that TNF-α
levels were significantly elevated in the exudates (Cuzzocrea
et al., 1999). In our results, we observed that non-stimulated
macrophages as well as macrophages stimulated by LPS
produce almost the same quantities of TNF-α; however
after in vivo stimulation by CAR we found a slight increase
of 30–40% in the cytokine release in vitro as compared to
the other groups.
In summary, we observed that cells stimulated with carra-
geenan showed a number of characteristics of activated
cells, such as altered morphology due to a greater number of
membrane projections and a greater number of vacuoles as
compared to non-stimulated macrophages as well as to mac-
rophages from mice stimulated in vivo with LPS. The bio-
chemical (high release of inflammatory mediators) data in
the present report also argue for the role of CAR as a potent
Table I. MEASUREMENTS OF NO, PGE2 AND TNF-α IN THE SUPERNATANTS COLLECTED FROM NON-STIMULATED, LPS- AND
CAR-STIMULATED CULTURES OF MICE PERITONEAL MACROPHAGES in vivo
NO (µM)PGE2 (ng/ml)TNF-α (ng/ml)
Macrophage Activation by CAR and LPS
inflammatory agent and recruiter of monocytes and mac-
rophages. Further biochemical studies are presently under
way to better clarify this activation induced by carrageenan.
Acknowledgments. We would like to thank Bruno Ávila for help with
image processing. This investigation was supported by FAPERJ, CNPq
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(Received for publication, January 20, 2004
and accepted, April 15, 2004)