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Departamento Sanidad Animal, Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria, Valdeolmos, Madrid, Spain; and
Servicio de Química de Proteínas, Centro de Biologia Molecular Severo Ochoa, Universidad Autónoma Madrid, Cantoblanco, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1), but antiinflammatory IL-10 is only detected on
the latter population. When 2A10- monocytes were cultured
in medium containing pig serum, they acquired some phenotypic features
of 2A10+ cells, expressing the 2A10 Ag. In contrast, when
they were cultured in the presence of L929 supernatant as a source of
GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over
basal levels. 2A10+ cells cultured with pig serum developed
features that resemble monocyte-derived dendritic cells. These results
indicate that 2A10+ monocytes could constitute a cell
population in a more advanced maturation stage than 2A10-
circulating monocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages originate from precursor cells in the bone marrow which sequentially develops into monoblasts, promonocytes, and monocytes. Monocytes enter the blood circulation from which they migrate to the various tissues, where they undergo further differentiation transforming into exudate macrophages. The maturation process ends when these exudate macrophages turn into resident macrophages. There is also evidence that some tissue resident macrophages are renewed by local proliferation. In vitro differentiation of peripheral blood monocytes in the presence of serum has been extensively used as a model system to investigate macrophage development (4). These studies have evidenced the high plasticity of the monocyte/macrophage lineage, since other cell types, in addition to macrophages, may be obtained from monocytes depending on the culture conditions (i.e., dendritic cells (DC)4 in the presence of GM-CSF and IL-4) (5).
The expression of surface markers has been widely used to identify different maturation stages within a particular lineage and to evaluate lineage-relationships among different cell types. In the human system, two major subsets of monocytes with distinct functional properties have been defined based on the expression of CD14, CD16, and CD64 Ags. Compared with CD64+CD14++CD16- cells, CD64low/-CD14+CD16+ cells express higher levels of MHC class II Ags and adhesion molecules (6, 7). CD16+ monocytes were also reported to have a lower phagocytic activity and capacity of production of oxygen radicals than CD16- monocytes (7, 8). Both subsets also differ in their accessory cell capacity and in their pattern of cytokine expression following LPS stimulation (7, 9, 10). All these findings have led to consider CD16+ monocytes in a more mature stage than CD16- cells (11).
The study of the swine immune system has been subject of considerable interest because of its importance in the design of better vaccines for controlling infectious diseases in this economically important livestock species. This interest has been spurred in the last years by the advances on xenograft transplantation and the potential use of this species as a source of organs. However, studies on the swine monocyte/macrophage lineage have been limited by the lack of specific markers. Although a significant number of mAbs have been raised against porcine mononuclear phagocytes, most of them are not lineage specific or recognize only a small subpopulation of macrophages. However, we have produced recently a panel of mAbs that recognize molecules whose expression is restricted to swine macrophages. One of these mAbs, named 2A10, seems to be of potential utility for studying the process of macrophage differentiation in swine. This mAb reacts with a 120-kDa monomeric glycoprotein, which is strongly expressed by the majority of tissue macrophages and at lower levels by a subset of monocytes (12).
In the present study, we have examined the heterogeneity of blood monocytes based on 2A10 expression with respect to the coexpression of surface molecules that are critical for effective monocyte function and in terms of their capacity as accessory cell to induce MLR and synthesize cytokines. Amino acid sequence analysis of peptides derived from the 2A10 molecule shows a high homology with the human M130 Ag, classified as CD163 at the 6th Human Leukocyte Typing Workshop (13). Our results indicate that 2A10+ monocytes correspond to mature stages of these cells and share some characteristics with differentiated macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Large-White outbred pigs were used as cell donors. Porcine alveolar macrophages (PAM) were collected by alveolar lavage as has been described (14). Swine PBMC were isolated on Percoll discontinuous gradients after blood sedimentation in dextran as described by González et al. (15). Bone marrow cells were obtained by perfusing ribs with PBS. Red cells and nonviable cells were removed by centrifugation at 400 x g for 10 min on lymphocyte separation medium at 1.077 g/ml (BioWhittaker, Walkersville, MD). For long-term cultures, monocytes were placed on Teflon jars at 2 x 106 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 5 x 10-5 M 2-ME, 20 mM HEPES, 30 µg/ml gentamicin (complete culture medium), and either 20% pig serum or 20% L929 cell culture supernatant (as a source of GM-CSF) (16).
Abs
Murine anti-swine leukocyte Ag mAbs 2A10/11 (IgG1), BL1H8
(IgG2b, CD11a), 2F4/11 (IgG1, CD11b), and 1F12
(IgG2b, swine histocompatibility leukocyte Ag (SLA) II DR)
were produced in our laboratory. Human CD14 mAb TÜK-4
(IgG2a) was obtained from Dako (Glostrup, Denmark). Human
4 integrin mAb HP2/1 (IgG1) was kindly provided by F.
Sánchez-Madrid (Universidad Autónoma Madrid, Madrid,
Spain). Swine CD16 mAb G7 (IgG1) and SWC9 mAb PM18/7 (IgG1) were a gift
from Y. B. Kim (Finch University, Chicago, IL). SWC1 mAb 76-6-7
(IgM) and SWC3 mAb 74-22-15 (IgG1) were kindly supplied by J. Lunney
(U.S. Department of Agriculture, Beltsville, MD). Characteristics of
the mAbs are summarized in Table I
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A total of 5 mg of purified 2A10 mAb were coupled to 1 ml of cyanogen bromide-activated Sepharose 4B beads (Pharmacia, Uppsala, Sweden), according to the indications of the manufacturer. PAM lysates were obtained from 3 x 109 cells. Lysis was performed in 1% Nonidet P-40, 50 mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, 0.1 mM PMSF, 10 µg/ml aprotinin, and 10 mM iodacetamide (lysis buffer) at 108 cells/ml for 1 h at 4°C. Lysates were precleared by incubation for 24 h with normal mouse IgG-coupled Sepharose beads. Then, precleared lysates were incubated for additional 24 h with 2A10 mAb-coupled beads. After that, beads were washed several times with lysis buffer and, finally, with PBS. The adsorbed fraction was eluted from the beads by adding 50 mM ethilenamine, pH 11. The eluted fraction was dialyzed against 10 mM ammonium bicarbonate, pH 8, and concentrated by lyophilization. Then, it was subjected to SDS-PAGE in 12% polyacrylamide and electrotransferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore, MA, USA). The main band, with an apparent molecular mass of 120 kDa, was excised and subjected to automated Edman degradation to obtain the N-terminal sequence. Internal peptides were obtained from protein digestion with trypsin, separated by HPLC, and also subjected to Edman degradation on an Applied Biosystem 473-A pulse liquid phase protein sequencer (Applied Biosystem, Foster City, CA). Peptide identities were searched on SwisProt, EMBL, and GeneBank databases using the GCG analysis program (17).
Immunofluorescence assays
Immunofluorescence (IF) analyses were performed with 106 cells (per Ab) on PBMC and bone marrow cells or with 105 cells when monocytes and PAM were used. For single-color IF, cells were incubated with 50 µl of hybridoma supernatant for 30 min at 4°C. Then, cells were washed with PBS containing 0.1% BSA and 0.01% NaN3 (fluorescence buffer) and incubated with FITC-conjugated goat F(ab')2 anti-mouse Ig (Dako) for 30 min at 4°C. Cells were then washed in fluorescence buffer, fixed in 0.1% paraformaldehyde, and analyzed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA). For two-color IF, after labeling with the first mAb as described above, cells were incubated with a biotin-labeled second mAb for 30 min at 4°C. Then, cells were washed again and incubated for 20 min at 4°C with PE-conjugated streptavidin (Southern Biotechnology Associates, Birmingham, AL). After washing, cells were fixed and analyzed as described above. Before analysis, unstained control cells and FITC- and PE-stained cells were run to set proper compensation and to define quadrants.
Sorting of 2A10+ and 2A10- monocytes
Blood monocytes were magnetically isolated by using the VarioMACS cell sorting technique (Miltenyi Biotec, Bergisch-Gladbach, Germany). Briefly, 5 x 108 PBMC were incubated with 5 ml of 2A10 hybridoma supernatant for 45 min on ice. Then, cells were washed with PBS containing 5% FCS and 2 mM EDTA (magnetic cell separation system (MACS) buffer) and incubated with 1 ml of goat anti-mouse IgG magnetic microbeads for 15 min on ice. After washing with MACS buffer, PBMC were passed through a MACS separation column (VS+ positive selection column) and magnetically labeled cells (2A10+ fraction) were collected. The effluent negative fraction was then incubated with 5 ml of 74-22-15 (anti-SWC3) hybridoma supernatant and MACS microbeads as described above. PBMC were passed through the separation column, and 2A10-SWC3+ cells were harvested (2A10- fraction). Isolated cells were left in complete medium for 12 h before used.
MLR assays
A constant number of 3 x 105 allogeneic PBMC were incubated with graded numbers of magnetically isolated 2A10+ or 2A10- monocytes. Monocytes were irradiated (3000 rad in a 137Cs source) and then cocultured with allogeneic PBMC for 5 days in 10% pig serum supplemented medium. Experiments were performed in 96-well plates in triplicate. T cell proliferation was measured by [methyl-3H]TdR (Amersham, Little Chalfont, U.K.) incorporation on day 5 of culture during the last 16 h. After freezing and thawing of the microplate cultures, the cells were harvested onto filters by using a multiharvester system (Tomtec, Orange, CT), and [3H]thymidine uptake was subsequently measured in a microplate scintillation counter (Wallac, Turku, Finland).
Cytokine detection
A total of 2 x 106 2A10+ and
2A10- isolated monocytes were incubated for 6 h on
6-well plates with or without LPS at 1 µg/ml. Then, total RNA was
extracted by using 100 µl/106 cells of Tripure isolation
reagent (Boehringer Mannheim, Indianapolis, IN) based on the method
described by Chomczynski and Sacchi (18). First strand cDNA was
prepared from 5 µg of total RNA, previously denatured by heating 2
min at 65°C and immediately placed on ice, with 5 µl of reverse
transcriptase (RT) reaction mix containing 10x Moloney murine leukemia
virus RT buffer (Epicenter, Madison, WI), 10 mM DTT, 0.5 mM oligodT, 50
µM each of dNTPs, 12.5 U Moloney murine leukemia virus RT
(Epicenter), and 20 U RNasin (Promega, Madison, WI). The reaction
mixtures were made up to 50 µl with RNase free water and incubated
for 1 h at 37°C. For PCR, a variable amount of the cDNA
(typically 2.5 µl) was used in a total volume of 25 µl of PCR mix
containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.005% Tween 20, 0.005%
Nonidet P-40, 0.5 or 1 mM MgCl2 (as empirically determined
for the different oligonucleotide pairs), 50 µM each of dNTPs, 10
pmol each of specific oligonucleotides (forward and reversed primers),
and 1 U Dnazyme II DNA polymerase (Finnzymes Oy, Helsenki, Finland).
PCR amplification was conducted for 30 cycles for TNF-, IL-10, and
IL-1, and 40 cycles for GAPDH of denaturation at 94°C for 45
s, annealing at 55°C for 30 s, and extension at 72°C for 1
min. PCR products were electrophoresed on 2% agarose gels containing
ethidium bromide and analyzed by an image densitometer coupled to a
computer program (Gelstation, TDI, Madrid, Spain). The primer sequences
and the expected fragment sizes in base pairs are shown in Table II. The GAPDH expression was used as
control for RNA content and integrity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The N-terminal sequence of 2A10 purified Ag was
Gly-Lys-Asp-Lys-Glu-Leu-Arg-Leu-Thr-Gly (Table III). This sequence showed an identity of
70% with the N-terminus of human M130 Ag, which is structurally
related to the scavenger receptor superfamily (19). Sequence comparison
of 2A10 Ag with N-terminus from other scavenger receptor cysteine-rich
(SRCR) domain-containing proteins showed lower homologies: i.e., 40%
to human CD5 (20), CD6 (21), and bovine WC1.1 Ag (22), 30% to bovine
CD5 (23), and 20% to the sea urchin speract receptor (24).
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). An identity of
62.5% was observed between peptide S38 and two distinct sequences from
the third and eighth SRCR domain of M130 Ag. Peptide S7, considering
the cysteine on the fifth position, shared 66.6% of identity with a
peptide from the M130 seventh SRCR domain. Finally, peptide S8 showed a
degree of identity of 87.5% with an internal sequence on the M130
eighth SRCR domain. No other sequences included on the SwisProt or
GeneBank databases showed a significant homology to the swine 2A10 Ag
peptides. 2A10 Ag expression on porcine myeloid cells
The distribution of 2A10 Ag has been previously shown to be
restricted to the swine monocyte/macrophage lineage, with the highest
levels of expression found in tissue macrophages (12). Here, we
compared the 2A10 expression on cells that can be considered as three
different maturation stages along the macrophage differentiation
pathway: bone marrow myeloid precursors, monocytes, and PAM (Fig. 1). Because of the lack of specific
markers for hemopoietic precursors in the swine, bone marrow myeloid
precursors were selected by the expression of SWC3 Ag. This molecule is
highly expressed along all stages of the myeloid differentiation
pathway (25). Monocytes were selected from PBMC on the basis of their
SWC3 expression and by their light scatter characteristics. PAM were
obtained from broncoalveolar lavage, and >95% of resultant cells bore
the SWC3 marker. As shown in Fig. 1, the expression of 2A10 Ag
increased throughout the maturation of the macrophage lineage. Thus,
while 2A10 molecules were practically undetectable on freshly isolated
bone marrow cells, almost 100% of PAM were positive for this marker.
In monocytes, two subpopulations could be distinguished, one negative
and the other expressing medium levels of 2A10 Ag. The percentage of
2A10+ monocytes varied among animals, ranging between
5–50% (data not shown).
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The above data prompted us to further characterize the phenotype
exhibited by monocytes with regard to the 2A10 Ag expression. Thus,
freshly isolated PBMC were double-stained with mAb 2A10 vs mAbs against
CD14, CD16, SLA II, SWC3, SWC9, CD11a, CD11b, and very late Ag-4
(VLA-4) markers (Fig. 2). Monocytes were
gated from PBMC by their light scatter properties. Results showed
several phenotypic differences among 2A10+ and
2A10- monocytes. 2A10+ cells expressed higher
levels of MHC class II molecules (SLA II) than 2A10-
monocytes. Expression of CD16, CD11a, and VLA-4 markers was also higher
on 2A10+ cells, although the differences with
2A10- cells were not so pronounced as in the case of SLA
class II. It is worthy to note that the whole population of porcine
monocytes bears the CD16 marker, in contrast to human monocytes. On the
other hand, the 2A10+ population showed lower levels of the
CD14 receptor than 2A10- monocytes. No major differences
were found between both cell subsets on SWC3 and CD11b expression. None
of the monocytes subpopulations were positive for SWC9, CD1, CD3, CD4,
CD8, IgM, and CD45RA markers (data not shown).
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2A10+ and 2A10- monocytes were
magnetically isolated to evaluate their morphological and functional
characteristics. 2A10+ monocytes were first isolated from
PBMC by mAb 2A10 binding. Then, 2A10- monocytes were
selected from the remaining cells by their reactivity with
anti-SWC3 mAb 74-22-15. When analyzed by light microscopy, both
cell subpopulations showed typical features of monocytes (Fig. 3A), with heterogeneous nuclei
of lobulated, reniform, or round shape and dense clumps of chromatin.
The 2A10+ cells were slight but significantly larger, with
an average size of 16.6 ± 1.6 µm over 14.8 ± 1.8 µm of
2A10- monocytes (p < 0.01). Fig. 3B shows the forward light scatter characteristics of
2A10+ and 2A10- cells 12 h after
isolation and a perceptible difference in their scatter parameters
(higher for 2A10+ cells) could be observed.
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Because the phenotype of 2A10+ monocytes appeared to
be reminiscent of that of mature tissue macrophages, we tested next
whether isolated 2A10- monocytes could differentiate and
express a similar surface phenotype after culture with either 20% pig
serum or 20% L929 cell-conditioned medium. Both media have been shown
to support the maturation of cultured swine monocytes (26, 27). The
results were compared with those obtained from 2A10+
monocytes under the same culture conditions. Fig. 4 shows the number of viable cells of
these subsets along a 5-day culture with homologous serum or L929
supernatant. We found no major variation of 2A10- cell
numbers on both conditions. In contrast, we observed larger differences
on cultured 2A10+ monocytes, with a pronounced cell death
in the cultures with pig serum, and a detectable increase of viable
cells in those with L929 supernatant.
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). On
2A10- monocytes, a decrease of CD14 and SWC1 surface
levels was observed after 5 days of culture with pig serum, together
with a slight increase of CD16 and SLA II Ags and a higher induction of
2A10 and SWC9 expression (up to 10 times over 12 h culture
levels). In contrast, on 2A10- cells maintained with L929
supernatant, only a small increase of 2A10, and none of SLA II and SWC9
was observed along the time of culture, although a similar reduction of
CD14 and SWC1 levels did occur. Likewise, culture of 2A10+
monocytes with pig serum led to an increased expression of SLA II,
2A10, and SWC9 and a reduction of CD14 and SWC1 expression. Finally,
2A10+ monocytes cultured with L929 supernatant showed a
reduction of CD14, SWC1, and SLA II levels, with only an small
induction of SWC9 expression on these cultures.
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2A10+ monocytes became also morphologically similar to
macrophages when cultured with L929 supernatant, but after culture with
homologous serum, they developed an stellate morphology with
cytoplasmic projections (Fig. 6). This last cell population was not
homogeneous; a small percentage of cells showed typical macrophage
morphology (not shown).
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Because of the different phenotype exhibited by 2A10+
and 2A10- monocytes, particularly with regard to the SLA
II molecules expression, we tested the ability of both subpopulations
to act as accessory cells in an allogeneic MLR. Thus, different amounts
of irradiated 2A10+ and 2A10- monocytes were
incubated with a constant number of allogeneic PBMC, and the
lymphoproliferative response was measured at day 5 of culture (Fig. 7). At lower cell concentrations
(6,000–12,000 cells/well), 2A10+ monocytes showed to be up
to 3–4 times more potent accessory cells than 2A10-
monocytes, whereas at the highest cell concentration tested (25,000
cells/well), both populations induced a similar response.
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Magnetically isolated 2A10+ and 2A10-
monocytes (>95% purity) were cultured for 6 h with or without
LPS at 1 µg/ml. Then, total RNA was extracted and a reverse
transcription followed by PCR was performed using specific primers for
TNF-, IL-1, IL-10, and the housekeeping gene GAPDH. Fig. 8 shows the results of a representative
experiment. mRNA for the proinflammatory cytokines tested, TNF- and
IL-1, was clearly induced upon LPS stimulation in both
2A10+ and 2A10- cells. Basal levels of these
cytokines varied among experiments. In contrast, mRNA induction for the
antiinflammatory cytokine IL-10 was only detected after LPS stimulation
of 2A10- monocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptors CD16, CD64, CD14, CD43) (7, 8, 30) is strictly specific of the monocyte/macrophage lineage. In this
study, we define two subpopulations of swine blood monocytes based on
the expression of a recently reported marker, the Ag recognized by mAb
2A10 (12), whose distribution is restricted to the
monocyte/macrophage lineage. 2A10 Ag can be considered as the porcine orthologue of human M130 Ag (19), recently assigned to CD163 at the 6th Human Leukocyte Typing Workshop, as peptide sequence identity between both Ags (62–87%) is within the range of interspecies molecular homology. Structurally, human M130 belongs to the scavenger receptor superfamily (19), and it contains nine SRCR domains. The SRCR domain has about 110 amino acid residues with six to eight conserved cysteines involved in intradomain disulfide bonds (31). Members of this ancient superfamily of proteins have been found from the lowest phylum of Metazoans (32) to mammals (19, 20, 21, 22, 23), and most of them seem to be involved in host defense (31). Despite of the conservation of SRCR domains along the evolution, human M130 Ag and no other reported proteins of the same superfamily showed significant similarity to peptides of the porcine 2A10 molecule.
Additional data support the identity between M130 and 2A10 Ags. Both markers seem to be specific of the monocyte/macrophage lineage, and no expression on other myeloid cells has been observed. Tissue distribution of both molecules is very similar; they are strongly expressed by macrophages of the red pulp of the spleen, the interfollicular and sinus macrophages of lymph nodes, the cortical macrophages of the thymus, and the Kupffer cells of the liver, while no reactivity is found on macrophages in follicles and the marginal zone of the spleen (12, 33, 34, 35). In our study, porcine 2A10 Ag appears to be a differentiation marker of the macrophage lineage; it is not detectable on freshly isolated bone marrow cells, being expressed at intermediate levels on blood monocytes and at high levels on alveolar macrophages. Likewise, M130 expression increases along the macrophage differentiation pathway, being weak on blood monocytes and strong on tissue macrophages. Furthermore, monoblastic (U937) and myelomonocytic (HL60) cell lines are negative for this marker (33, 34). On the other hand, the apparent molecular mass reported for M130 Ag varies from 130 kDa to 140 kDa, depending on the authors (33, 35); whereas 2A10 Ag shifts from 120 kDa before reduction to 150 kDa after reduction, this feature is indicative of the presence of intrachain disulfide bonds (12) and agrees with the characteristics of SRCR domains. Finally, although these molecules are expressed on the cell surface, substantial amounts of both of them are located intracellularly. Cloning of the 2A10 Ag is currently in progress, which will aid to define its homology with M130 molecule.
The two subpopulations of swine monocytes defined on the basis of the expression of 2A10 Ag phenotypically resemble the human monocyte subsets CD14++CD16- (2A10- monocytes) and CD14+CD16+ (2A10+ monocytes) (6, 8, 11). In swine, the whole population of blood monocytes bears the marker CD16, but 2A10+ monocytes express it at higher levels than 2A10- cells. Moreover, CD14 expression is clearly diminished on 2A10+ in relation to 2A10- monocytes. Additional markers such as SLA II, VLA-4, and LFA-1 are increased on 2A10+ monocytes, like on human CD16+ monocytes. No significant differences on CD11b levels were observed between 2A10- and 2A10+ monocytes, although its expression is decreased on human CD16+ monocytes (6, 36). In fact, unlike human CD11b, whose expression is confined to well-differentiated myelomonocytic cells (37, 38), swine CD11b is expressed on almost 100% of SWC3+ bone marrow cells, and its level is maintained along the stages of differentiation of the swine monocyte/macrophage lineage (data not shown).
Both porcine 2A10+ and human CD16+ monocyte
subsets represent a minor percentage within the circulating monocyte
population (8). Interestingly, both subsets fail to produce IL-10 in
response to LPS stimulation (10). IL-10 is one of the best known
inhibitory cytokines of the monocyte/macrophage activation (39). Its
role as an antiinflammatory factor is well documented, as it inhibits
the synthesis of IL-1, IL-6, TNF-, and GM-CSF on LPS-stimulated
monocytes (40). Furthermore, it has an inhibitory effect on IFN-
production by Th1 cells, partly through the inhibition of IL-12
synthesis by accessory cells, which contributes to the suppression of
cellular responses (41). The absence of IL-10 production by human
CD16+ monocytes has been interpreted as though these cells
are of a proinflammatory type (10). Their high levels of LFA-1 and
VLA-4 integrins (6) support this hypothesis, because it might allow a
rapid monocyte transvasation from the blood to the inflamed tissues.
Both characteristics are also present on 2A10+ monocytes,
which have been also shown to produce inflammatory cytokines (TNF-
and IL-1) after bacterial LPS stimulation.
However, there are some significant differences between swine 2A10+ and human CD16+ monocytes. Human CD16+ monocytes are smaller and denser cells than CD16- monocytes, whereas 2A10+ are slightly larger than 2A10- monocytes. Further differences come from functional studies on CD16+ monocytes. Thomas and Lipsky (9) reported a low accessory and APC capacity for this human monocyte subset. In contrast, our results show that 2A10+ monocytes are strong stimulatory cells in MLR, which is in accordance with the data reported by Grage-Griebenow et al. on human CD64- monocytes (7). These cells share phenotypical characteristics with swine 2A10+ and human CD16+ monocytes. Isolation procedures might be the cause of such differences, as the three populations have been separated by sorting with distinct mAbs. However, based on those morphological and functional discrepancies, we cannot discard that 2A10+ monocytes represent a subpopulation different from human CD16+ monocytes.
Compared with 2A10- monocytes, the phenotype of 2A10+ monocytes is closer related to that of mature macrophages. Thus, PAM express high levels of 2A10, CD16, and SLA II molecules, whereas their CD14 levels are lower than those of 2A10- monocytes (data not shown). On the other hand, as has been mentioned before, 2A10+ monocytes express higher levels of the integrins LFA-1 and VLA-4, appearing to be better endowed to leave the blood and reach the tissues. Therefore, they might represent the precursors of monocyte-derived tissue macrophage populations.
When magnetically isolated 2A10- monocytes were cultured in vitro with porcine serum, they developed a phenotype close to that of 2A10+ monocytes, reinforcing the idea that both subsets represent two different stages of maturation. On those cultured cells, the expression levels of 2A10, CD16, and SLA II Ags increased, but CD14 expression diminished. However, the levels of tested integrins (LFA-1, CD11b, and VLA-4) did not show significant changes during the culture (not shown). Two markers of swine macrophage differentiation, SWC9 and SWC1, were also analyzed. Within the porcine monocyte/macrophage lineage, SWC9 is expressed exclusively on mature macrophages, while SWC1 expression is remarkably higher on monocytes than on tissue macrophages (42). As expected, serum-induced monocyte maturation was accompanied by an increment of SWC9 and a reduction of SWC1 expression, both in 2A10- and 2A10+ cells. 2A10- monocytes cultured with L929 supernatant showed an intermediate phenotype, particularly in relation to 2A10 and SWC9 Ags, whose up-regulation seems to be mostly dependent on factors present in the serum.
One striking feature of 2A10+ monocytes is the morphology that they acquire after a few hours of culture with pig serum. On these conditions, while 2A10- monocytes are driven to a macrophage morphology, 2A10+ cells develop features that closely resemble monocyte-derived DC (43). It is worthy to note that these cells were grown on suspension cultures, therefore the cytoplasmic projections that they develop cannot be attributed to adherence processes in a solid substrate. Functional DCs can be generated from purified human blood monocytes after GM-CSF plus IL-4 treatment, and their morphologic changes (43) are similar to those observed in serum-cultured 2A10+ monocytes. Unfortunately, most of the markers employed to characterize DCs (i.e., CD40, CD83, CD86) are unavailable in the porcine system. However, the up-regulation of MHC class II and the loss of CD14 expression that we find on serum-cultured 2A10+ monocytes are characteristics of human monocyte-derived DCs (43, 44). Furthermore, preliminary results reveal that CD1, a MHC class I-like molecule whose expression is induced on PBMC-derived DCs (43, 45), is expressed on the membrane of 2A10+, but not of 2A10- monocytes (not shown). As mentioned above, isolated 2A10+ monocytes possess a great stimulatory capacity in MLR, which could be correlated, at least in part, with their high expression of SLA II and the costimulatory molecule LFA-1 (46). This capability is shared by DCs (45), although the allogeneic T cell proliferation induced by those cells is frequently stronger than that reported here for 2A10+ monocytes. Altogether, our findings indicate that, although freshly isolated 2A10+ blood cells have phenotypic and morphologic features of monocytes, they can be driven under certain conditions to cells with some characteristics of DCs. Studies on swine DCs isolation and phenotyping will help us to understand their relationship with 2A10+ monocytes.
In summary, we have characterized a new subpopulation of swine monocytes based on the expression of a macrophage differentiation marker, the 2A10 Ag. This subpopulation presents some features of mature tissue macrophages and inflammatory cells. These cells could also be in vitro precursors of DCs, because they share specific morphological and functional properties characteristic of those accessory cells.
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Acknowledgments |
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Footnotes |
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2 Current address: CINVESTAV, Av IPN2508, S. Pedro Zacatenco, C.P. 07300, Mexico.
3 Address correspondence and reprint requests to Dr. J. Domínguez, Departamento Sanidad Animal, Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria Valdeolmos 28130, Madrid, Spain. E-mail address:
4 Abbreviations used in this paper: DC, dendritic cells; PAM, porcine alveolar macrophage; IF, immunofluorescence; SLA, swine histocompatibility leukocyte Ags; SRCR, scavenger receptor cysteine-rich; MACS, magnetic cell separation system; RT, reverse transcriptase; VLA-4, very late Ag-4.
Received for publication November 9, 1998. Accepted for publication February 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor I (CD64)-negative monocytes, a minor human monocyte subpopulation with high accessory and antiviral activity. Eur. J. Immunol. 23:3126.[Medline]
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  H. Moriyama, M. Kobayashi, T. Takada, T. Shimizu, M. Terada, J.-I. Narita, M. Maruyama, K. Watanabe, E. Suzuki, and F. Gejyo Two-dimensional Analysis of Elements and Mononuclear Cells in Hard Metal Lung Disease Am. J. Respir. Crit. Care Med., July 1, 2007; 176(1): 70 - 77. [Abstract] [Full Text] [PDF]
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