Rosette formation by macrophages with adhered T lymphocytes is precluded by inhibitors of antigen processing and presentation
Ivón Teresa Novak and Humberto Ramón Cabral
Instituto de Biología Celular, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Av. Enrique Barros, Ciudad Universitaria, 5000 Córdoba, Argentina.
Address correspondence to: Dr. Humberto R.A. Cabral. Instituto de Biología Celular, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, ARGENTINA. E-mail: hcabral@fcm.unc.edu.ar
ABSTRACT: We had previously found in autologous human leukocyte cultures, in which dead neutrophils phagocytosis by macrophages occur, macrophages and T CD4 lymphocytes perform a selective cell-cell interaction showing many figures of either one, two or several T- lymphocytes adhering to a central macrophage were seen. Considering that antigen presentation would be necessary for the formation of these immune synapses, we attempted to block rosette formation (i.e., the formation of macrophage associations with at least three lymphocytes) by interfering with both antigen processing and presentation. Culture samples of autologous leukocytes from 7 healthy donors were subjected to either brefeldin A, chloroquine or to an anti- HLA DR antibody. Rosette formation was significantly inhibited in the treated samples (either with brefeldin A, chloroquine or the anti- HLA DR; ANOVA, p<0.001, as compared with the untreated controls). It is concluded that interference with antigen processing and presentation precludes the formation of these cellcell interactions.
Key words: Autologous rosetting; Cultured human macrophages and T- lymphocytes; Brefeldin A; Chloroquine; Immune synapses.
Introduction
One immune synapse is formed when a T lymphocyte
adheres closely to an antigen-presenting cell
(Grakoui et al., 1999; Lee et al., 2002; Dustin, 2005).
We found that immune synapses occur in cultures of
autologous human leukocytes, in which phagocytosis
of dead neutrophils occurs, and in which rosettes of
one macrophage surrounded by lymphocytes occur
(Cabral and Novak ,1992). Single immune synapses or
rosettes first appeared after 15 h in culture (Cabral and
Novak, 1992, 1999), and this phenomenon was dependent
on both time and cell density. Immunocytochemically,
the lymphocytes surrounding macrophages were
T CD4+ and they also positively reacted for UCHL-1,
indicating that they were activated T-cells (Cabral and
Novak, 1999).
We have previously observed that co-culture of both
purified macrophages and lymphocytes did not induce
rosette formation unless antigens were added to the culture
medium (Cabral and Novak, 2003). We postulated,
therefore, that antigen presentation would be involved
in the formation of such immune synapses and rosettes
in autologous leukocyte cultures, because an antigenic
stimulus would derive from dead neutrophils in culture,
whose materials (such as CD66 antigen) were identified
inside monocyte-derived macrophages (Cabral and
Novak, 1992, 1999; Novak and Cabral, 2002).
Insofar as cell-cell interactions in rosettes may correspond
to an interaction of T CD4+ cells with class II
major histocompatibility complex (MHC) molecules
expressed by macrophages, the current study was
launched to study the effects of brefeldin A and chloroquine (inhibitors of antigen-proccesing and presentation)
as well as of a monoclonal antibody against anti-HLA molecules, respectively, on the immune synapse
and rosette formation, considering that if antigen presentation
is a necessary stimulus for the immune synapses
and rosettes production.
Brefeldin A is a metabolite of the fungus Eupenicillium
brefeldianum, which specifically and reversibly
blocks protein transport from the endoplasmic reticulum
to the Golgi apparatus (Klausner et al., 1992; Pelham,
1991), and was first described as a specific inhibitor of
antigen processing for Class I-restricted T cell recognition
(Yewdell and Bennink, 1989). However, currently,
the inhibition of phagocytic antigen processing by
brefeldin A has been described and correlated with depletion
of nascent MHC-II molecules in phagosomes
(Ramachandra and Harding, 2000). Chloroquine is known
to increase the pH of endocytic vacuoles, both inhibiting
antigen processing and presentation in the phagocytic
pathway (Janeway et al., 2004). With respect to the use
of anti-HLA DR, the idea was to block the HLA-DR
molecule at the surface of the antigen presenting cells
(the monocyte-derived macrophages).
Materials and Methods
Autologous Leukocyte Cultures
Blood samples were obtained from normal adult
volunteers who gave their written informed consent (5
women, 4 men; mean age 32 years, range 18-58 years).Venous blood was obtained with sterile disposable syringes
containing phenol-free heparin. Blood samples
were allowed to sediment for 2 h. Plasma supernatants
containing all types of leukocytes were separated and
were cultured at 37ºC in TC199 medium (DIFCO, Detroit,
Michigan, USA) supplemented with penicillinstreptomycin
and 25% filtered autologous serum). Cell
concentration was 300.000 leukocytes/mL. Culture
samples were harvested with previous shaking of the culture
flasks and centrifuged at 200 g and cytopreparations
were made of each cell pellet, as follows: the cell pellets
were gently dispersed in drops of TC 199 culture
medium and distributed with Pasteur pipettes onto identified
glass slides and left to settle for 5 min, in a humid
chamber, and then desiccated by speedy rotation over a
disk fixed on the axis of a centrifuge (Cabral and Novak,
1992, 1999). Control experiments were leukocyte cultures
of the same donors, but in which brefeldin A, chloroquine
or anti-HLA-DR were not added. Cell viability
was assessed by the trypan blue exclusion test in all
samples.
Cytopreparations were stained with either May-Grünwal-Giemsa, hematoxylin-eosin or toluidin blue
and were studied under a bright field microscope. A
macrophage surrounded either by one or two lymphocytes
was called an immune synapse, while if it was surrounded by three or more lymphocytes was called a "rosette". The percentage of both immune synapses and
rosettes in a given cytopreparation was computed after
counting 100 macrophages.
Brefeldin A (SIGMA, St. Louis, MO) was added 4
and 7 h after the initiation of cell cultures and at either
5 µg/mL, 2.5 µg/mL or 1 µg/mL. In some experiments,
the cells were washed with filtered autologous serum at
4 h after the addition of brefeldin A, they were centrifuged
for 10 min at 200 g, and were cultured again in a
medium without brefeldin A. Chloroquine (Rhône-Poulenc) was added at the initiation of cell cultures,to
obtain a 0.1 mM concentration. The study was made at
several times (after 24, 48, 72, 96, 120, and 144 h). Cell
cultures without chloroquine were used as controls.
Anti-HLA
Venous blood was obtained from 4 human healthy donors. Autologous leukocyte cultures were performed as described above. A monoclonal antibody against HLA-DR (Diatec, Norway) was added to leukocyte cultures after 72 and 96 hours of culture, at a final concentration of 15 µg/mL, and was incubated for 30 minutes. Next, the technique for immune synapse and rosette expression was perfomed (Cabral and Novak, 1992, 1999). Cultures without anti-HLA-DR treatment were employed as controls. All samples were harvested and centrifuged at 200 g and prepared as described above.
Statistical analysis
Analysis of variance (ANOVA) and t test for paired differences were used for statistical evaluations.
Results
In the control experiments, rosettes were observed in cytopreparations from cell cultures after 15 hours. Figure 1A shows the harvested cells after 3 hours of culture, when nor rosettes were formed (Cabral and Novak, 1992, 1999), while figures 1B and 1C show rosettes formed at 48 and 72 hours of culture, respectively. The trypan blue exclusion test yielded values above 96% in all experiments.
FIGURE 1. (A) Cytopreparation of culture from autologous human leukocytes obtained after 3 h of culture. Note that the
cells are not forming any close association; hematoxylin-eosin, x200. (B) Rosette formed between a monocyte-derived
macrophage and lymphocytes in a 48 h culture of leukocytes from a healthy subject; May-Grünwald-Giemsa, x400 and
amplified. (C) Rosette formed between a human monocyte-derived macrophage and lymphocytes from a 72 h leukocyte
culture; May-Grünwald-Giemsa, x400. (D) Leukocyte culture with brefeldin A at 5 µg/ml where no rosettes were formed;
macrophages showed a spongy aspect; May-Grünwald-Giemsa, x400.
Experiments with BFA at 5 µg/mL
Rosettes were not formed when brefeldin A was added to these cell cultures (Fig. 2A). Monocytes already showed signs of their transformation into macrophages after 15 h of culture. After 48 h of culture with brefeldin A (Fig. 2A), macrophages showed a spongy aspect (Fig. 1D).
FIGURE 2. Macrophage-lymphocyte rosettes (MLR) in autologous leukocyte cultures with the addition of brefeldin A
(BFA). The results are expressed as the percentage of the number of macrophages that formed rosettes in a given
cytopreparation by counting 100 macrophages, (mean ± 2 SD). (A) 5 µg/mL in vitro; (B) 2.5 µg/mL in vitro and washed
at 4 h. The ANOVA showed significant differences in rosette formation (p<0.0001) between treatments (BFA at 2.5 µg/
mL in vitro, and without BFA); (C) 1 µg/mL in vitro, at 4 and 7 h; (D) 1 µg/mL in vitro, and washed at 4 h.
Experiments with brefeldin A at 2.5 µg/mL and washed at 4 h
The effect of brefeldin A on cell cultures was reversed by washing with autologous serum after 4 h of culture (Fig. 2B). In such conditions, macrophages conserved their morphology after 72 h. Only a low number of macrophages had a single adhered lymphocyte after 96 h of culture.
Experiments with brefeldin A at 1 µg/mL, observed at 0, 4, and 7 h of culturing
Rosettes did not occur when brefeldin A was added to cell cultures at 0, 4 or 7 h, which contrasted with rosette formation in control cultures in which no brefeldin A was added (See Fig. 2C).
Experiments with brefeldin A at 1 µg/mL and washing at 4 h
Samples of leukocyte cultures with and without brefeldin A were harvested at 24 h, 28 h, 52 h, 76 h and 100 h (Fig. 2D). Significant differences in rosette formation were found (ANOVA, p<0.0001) between brefeldin A treated cultures and the controls. As expected, the number of immune synapses and rosettes increased in cultures that were washed at 4 h. These results showed that brefeldin A inhibits macrophage activity and rosette formation.
Effect of chloroquine on rosette formation
Inhibition of rosette formation was observed under chloroquine treatment (Fig. 3), which contrasted with rosette formation in the control cultures with no chloroquine added.
FIGURE 3. Macrophage-lymphocyte rosette (MLR) formation
in autologous leukocyte cultures, with and without
chloroquine (0.1 mM) treatment in vitro. The results
are expressed as the percentage of the number of macrophages
that formed rosettes in a given cytopreparation
by counting 100 macrophages (mean ± 2 SD).
Effect of anti HLA-DR
The addition of anti HLA-DR to the cell cultures significantly inhibited the formation of both immune synapses and rosettes, both at 72 and 96 hours, as compared with the control cultures (Figs. 4 and 5; ANOVA, p<0.0001).
FIGURE 4. No rosettes nor immune synapses
were formed in autologous leukocyte
cultures treated with anti HLA-DR
(96 h of culture); toluidin blue, x400.
FIGURE 5. Macrophage-lymphocyte rosettes (MLR) in autologous
leukocyte cultures treated with an anti HLA-DR
monoclonal antibody. The results are expressed as the percentage
of the number of macrophages that formed rosettes
in a given cytopreparation by counting 100 macrophages,
(mean ± 2 SD). ANOVA showed significant differences in
rosette formation (p<0.0001) between treatments (with and
without the addition of HLA-DR antibody)
Discussion
Macrophage-lymphocytic rosettes. i.e., the occurrence
of several areas of close contact between a macrophage
and several adhered T lymphocytes was found
in previous work (Cabral and Novak, 1992, 1999) when
cells were sampled from autologous leukocytes cultures.
The central macrophage in them showed signs
of phagocytosis of dead neutrophil materials. In the
same cytopreparations many figures of one macrophage
with only one or two adhered lymphocytes were
found, thus, it would appear as an adequate model for
studying features of the formation of both individual
and multiple immune synapses between macrophages
and T lymphocytes.
In a previous study (Cabral and Novak, 2003) we
found that no rosettes were formed in co-cultures of
purified, autologous monocytes and lymphocytes unless
antigens were added to these cultures. In the current
experimental model, in which antigens from dead
neutrophils occur, both immune synapses and rosettes
occurred 15 hours after the beginning of cultures, when
blood monocytes showed morphological signs of macrophage
transformation and have ingested materials
from dead neutrophils (Cabral and Novak, 1992, 1999).
Moreover, we found that the number of rosettes
increased from 15 to 120 hours of culture (Cabral and
Novak (1992, 1999). The lymphocytes forming the rosettes
are T-CD4 cells and were also positively reacting
for CD45RO (clone UCHL-1), indicating that they are
activated T-cells (Cabral and Novak, 1999).
Here, we have experimentally explored the formation
of either single or multiple immune synapses and
rosettes, after subjecting the cultured leukocytes to
agents that could either prevent antigen-processing-presentation
(as brefeldin A or chloroquine), or may act on
the surface molecules of antigen presentation class II
alone (antibodies anti HLA DR).
In recent studies, the inhibition of phagocytic antigen
processing by brefeldin A have been described and
correlated with depletion of nascent MHC-II molecules
in phagosomes (Ramachandra and Harding, 2000). Our
results would reinforce this concept. On the other hand,
the finding that the number of rosettes increased in
samples of cultures washed at 4 h, suggests the
reversibility of effects of brefeldin A, as it was observed
in other experimental models (Klausner et al., 1992;
Pelham, 1991). In cell cultures with brefeldin A, macrophages
showed morphological changes (Fig. 1D), also
corroborating previous findings by other authors
(Klausner et al., 1992; Pelham, 1991). We were interested
in investigating the effects of brefeldin A at various
times of cell culturing, as well as at several times of
assays and conditions, because in this model, both rosettes
and immune synapses are observed since 15 h of
culturing, while brefeldin A effects have been reported
in short- time assays only (Lippincontt-Schwartz et al.,
1991; Hunziker et al., 1991). In our experiments, we
found that a brefeldin A concentration of 1 µg/mL is a
satisfactory concentration for these cellular studies.
Chloroquine has been described as an inhibitor of
antigen processing and presentation in the phagocytic
pathway, by increasing the pH of endocytic vacuoles
(Janeway et al., 2004).
The results of this work showed that both brefeldin
A and chloroquine inhibit the formation of immune synapses
and autologous rosettes between macrophages and
T lymphocytes. Exposure to an anti-HLA DR antibody
also precluded the formation of immune synapses and
rosettes. These results strongly suggest that both antigen
processing and presentation are involved in the occurrence
of immune synapses and rosettes. The effect
of anti-HLA DR treatment, also suggests that such antibody
can preclude the presentation at the molecular
level, at the surface of the antigen processing cell.
Hence, taken together, the current results support
the hypothesis that antigen presentation would be a necessary
stimulus for rosettes formation.
Acknowledgments
We thank Professor Martha Gonzalez Cremer for help in the preparation of the manuscript and Professor Silvia Joekes for statistical advice.
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Received on May 20, 2007.
Accepted on August 17, 2007.