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* Departments of Immunology and
Molecular Biology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City
Correspondence: Carlos Rosales, Department of Immunology, Instituto de Investigaciones Biomédicas–UNAM, Apto. Postal 70228, Cd. Universitaria, México D.F.–04510, Mexico. E-mail: carosal{at}servidor.unam.mx
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-), wortmannin and PD98059 blocked Fc receptor-mediated phagocytosis efficiently. Inhibition of phagocytosis by PD98059 was observed after 24 h of IFN- treatment, whereas wortmannin could inhibit phagocytosis only after 48 h of IFN- treatment. Additionally, phagocytosis of IgG-coated erythrocytes by neutrophils, a more efficient phagocyte, was inhibited by wortmannin and PD98059. Neutrophils and monocyte-differentiated macrophages presented significantly more efficient phagocytosis than monocytes upon PMA stimulation. Taken together, these results indicate that poorly phagocytic leukocytes, such as monocytes, do not require PI 3-K and ERK for phagocytosis. Upon differentiation into macrophages, however, ERK first and PI 3-K second are recruited for regulation of phagocytosis. In addition, our data support the idea that professional phagocytes require ERK and PI 3-K for efficient phagocytosis.
Key Words: signal transduction • luciferase • EIgG • insoluble immune complex
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R) on many cells of the immune system triggers various functions such as phagocytosis, antibody-dependent cell-mediated cytotoxicity, generation of the respiratory burst, and production of inflammatory mediators and cytokines [1
, 2
].
Three classes of FcR have been identified: FcRI (CD64), FcRII (CD32), and FcRIII (CD16) [3
]. After FcR aggregation and activation of Src and Syk family protein tyrosine kinases, several substrates are phosphorylated, and other enzymes are also activated. Among them, phospholipase C 1 and 2, phospholipase A2 (PLA2), paxillin, phosphatidylinositol 3-kinase (PI 3-K), and extracellular signal-regulated kinase (ERK), also known as mitogen-activated protein kinase, have all been reported [4
, 5
].
One of the major cellular responses initiated by FcR cross-linking, especially in myelomonocytic cells and in neutrophils (PMN), is phagocytosis [6
]. The molecular machinery needed for this function is of great interest and active research [7
]. Studies indicate that ERK is needed for phagocytosis of IgG-opsonized particles by PMN [8
, 9
]. However, other reports indicate that ERK is not required for phagocytosis by monocytes [10
, 11
]. So it seems that ERK may be involved in phagocytosis in some cases but not in others, depending on the cell type. Similarly, PI 3-K, a lipid kinase that phosphorylates phosphoinositides at the 3' position of the inositol ring [12
], has been reported to be an important molecule for regulation of FcR-mediated phagocytosis by professional phagocytes (PMN and macrophages) [13
14
15
16
]. However, we recently reported that in monocytes, FcR-mediated phagocytosis can proceed independently of PI 3-K [11
]. It thus seems that, like ERK, the participation of PI 3-K in phagocytosis is not general to all phagocytes.
These studies suggested that professional phagocytes (PMN and macrophages) present more efficient phagocytosis because they use PI 3-K and ERK for this function, whereas monocytes do not. To test the hypothesis that differentiation of monocytes into macrophages is accompanied by recruitment of PI 3-K and ERK to the phagocytic process, we assessed the participation of these signaling molecules in the phagocytosis of IgG-coated erythrocytes (EIgG) by monocytic cells and by monocyte-differentiated macrophages.
Data in this report support the idea that professional phagocytes require ERK and PI 3-K to accomplish their phagocytic functions and that these enzymes are recruited for regulation of phagocytosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RI (mAb 32.2), anti-FcRII (mAb IV.3), anti-FcRIII (mAb 3G8), and anti-major histocompatibility complex (MHC) class I (mAb W6/32) were from American Type Culture Collection (Manassas, VA). The specific PI 3-K inhibitors, wortmannin and LY294002, and the protein kinase C (PKC) inhibitor staurosporine were from Calbiochem (San Diego, CA). The specific MEK (ERK kinase) inhibitor PD98059 was from New England Biolabs (Beverly, MA). Recombinant human interferon- (IFN-) was from Endogen (Woburn, MA). The plasmid 3XMHC-luciferase (luc) contains nuclear factor-
B (NF-B)-responsive elements upstream of the luc reporter gene and has been described [4
, 17
]. All other chemicals were from Sigma Chemical Co. (St. Louis, MO).
Cell culture
The human monocytic THP-1 cell line was maintained at a density of 0.8 x 106 cell/ml in RPMI-1640 medium (Gibco-BRL, Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL), 20 µM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin.
Purification of neutrophils and monocytes
Neutrophils and monocytes were obtained from heparinized venous blood from healthy adult donors and purified by standard techniques as described previously [18
19
20
].
Opsonization of sheep erythrocytes
Sheep red blood cells were purchased from Erikar, S. A. (Mexico City, Mexico). IgG-opsonized erythrocytes (EIgG) were prepared as follows. Red cells were washed three times in Alsever’s solution (0.1 M dextrose, 40 mM citric acid, 10 mM NaCl, pH=6.1) and adjusted to a concentration of 1 x 109 cell/ml. This suspension (1 ml) was mixed with 0.9 ml Alsever’s solution and 100 µl of a 1/1000 dilution of rabbit serum anti-sheep erythrocytes. This mixture was incubated for 10 min at 37°C. Unbound antibody was removed by washing the cells several times with Alsever’s solution.
Phagocytosis
Phagocytosis of EIgG by monocytes, macrophages, and PMN in the fluid phase was performed as described previously [18
]. Phagocytosis was scored by light microscopy, counting cells at high maginification, and reported as phagocytic index (PI), that is, the number of EIgG ingested by 100 leukocytes. In assays involving inhibition of PI 3-K, ERK, or PKC, phagocytes were previously incubated with the corresponding inhibitor at the following concentrations: 50 nM wortmannin, 50 µM LY294002, 50 µM PD98059, or 2.5 nM staurosporine. In selected experiments, phagocytes were stimulated by addition of 100 ng/ml phorbol 12-myristate 13-acetate (PMA) throughout the phagocytosis assay.
Insoluble immune complexes (IIC)
IIC were prepared as described previously [4
, 19
], using 300 µl rabbit anti-horse ferritin serum and 30 µl horse ferritin type I (100 mg/ml; Sigma Chemical Co.).
FcR stimulation
Cells (1x107) in 5 ml serum-free RPMI-1640 medium were stimulated by addition of 40 µl IIC and incubation for 3 min at 37°C. Cells were then lysed in assay buffer as described [4
, 21
].
Western blot
ERK and PI 3-K were detected by immunoblotting with the corresponding antibody: anti-ERK mAb at 75 ng/ml, anti-PI 3-K at 50 ng/ml, or anti-pERK at 75 ng/ml as described [21
].
Transfections and FcR stimulation
THP-1 monocytic cells were transiently transfected using a (diethylamino)ethyl-dextran method as described previously [17
]. For FcR stimulation, cells were mixed with 40 µl IIC. Luc enzymatic activity was then determined as described [4
, 21
].
Monocyte to macrophage differentiation
THP-1 cells, at approximately 0.8 x 106 cell/ml, were treated with 1 µM retinoic acid for 48 h. Cells were then washed twice with medium, resuspended in fresh medium supplemented with 5% heat-inactivated FBS, and incubated with 150 ng/ml IFN- for an additional 24 or 48 h, as described previously [22
, 23
].
Determination of peroxidase
Cells (1.5x106) were lysed in 45 µl cold water with vigorous vortexing. After addition of 5 µl 10x phosphate-buffered saline (PBS), lysates were clarified by centrifuging 5 min at maximum speed in a microfuge. In 96-well plates, 50 µl cell lysates were mixed with 100 µl 0.2 M sodium phosphate, pH = 6.2, and 25 µl of a mixture of 2 mM diaminobenzidine and 7.5 mM H2O2. The mixture was left at room temperature for 10 min in the dark. Peroxidase activity, indicated by color intensity, was read in a microplate reader (Bio-Tek Instruments Inc., Winooski, VT) at 490 nm.
Kinase activity assay
ERK was immunoprecipitated from cell lysates, and its activity was determined as described [4
]. Similarly, PI 3-kinase was immunoprecipitated from cell lysates, and its activity was determined as described [21
]. Radioactivity associated with the phosphorylated products was quantitated using a phosphoimager (Molecular Imager FX, Bio-Rad, Hercules, CA).
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA
Total RNA was isolated by cesium chloride gradients from monocytes or monocyte-differentiated macrophages as described previously [24
]. Each RNA (3 µg), dissolved in 50 mM Tris-HCl, pH = 8.0, 70 mM KCl, 10 mM MgCl2, 1 mM each of the four triphosphate-deoxyribonucleosides, and 4 mM dithiothreitol, was transcribed with 200 units of avian myeloma virus RT (Boehringer Mannheim, Indianapolis, IN) using 0.5 µg oligo(dT)12–18 in a total volume of 50 µl at 42°C for 90 min. The cDNA was then purified by phenol-chloroform extraction, precipitated with ethanol, and resuspended in 50 µl water. The cDNA (1, 5, and 10 µl) was amplified by PCR with 30 cycles of 1 min at 94°C, 2 min at 55.5°C, and 2 min at 72°C. The primers used for amplification were RR-1: 5'-AAA CGG ATC ACA GTG GAG GAA GCG CTG GCT CAC CCC TAC C-3'; RR-2: 5'-GCA GGG GCG CCG GGC TCT CCA CGC CCC CCA GCT CCA CTT C-3' for the ERK1 gene; RR-3: 5'-AAT AAA CTT AAC ACA GAG GAA ACT GTA AAA GTT CAT GTC A-3'; and RR-4: 5'-TCA GAG AGG GCT TCC CGG TAA GCA CTC TGT TTT AAA CAG G-3' for the PI 3-K (p110 ß isoform) gene. These primers amplify an internal fragment of 779 bp for the ERK gene and an internal fragment of 1249 bp for the PI 3-K gene, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R-dependent phagocytosis by professional phagocytes [8
, 9
, 13
14
15
16
]. We decided to confirm these results measuring phagocytosis of EIgG by unstimulated neutrophils. These phagocytes are able to ingest nonopsonized erythrocytes presenting a basal PI of approximately 15 (Fig. 1
). IgG-opsonized erhythrocytes (EIgG) were ingested at a PI of approximately 30. This increment therefore represents phagocytosis mediated by Fc receptors. As expected, the PI 3-K inhibitors (wortmannin and LY294002) and also the MEK (ERK kinase) inhibitor (PD98059), which causes ERK inhibition indirectly, blocked it completely (Fig. 1A)
. Thus, in PMN, unstimulated, FcR-mediated phagocytosis depended on PI-3K and ERK, as expected.
|
R-mediated phagocytosisR-mediated phagocytosis at levels comparable with those of unstimulated neutrophils. THP-1 cells were allowed to ingest EIgG after incubating them with wortmannin, LY294002, or PD98059. None of these inhibitors had any effect on this type of phagocytosis (Fig. 1B)
. Similarly, peripheral blood monocytes presented normal phagocytosis levels in the presence of wortmannin, LY294002, or PD98059 (Fig. 1C)
. Additionally, the generic PKC inhibitor, staurosporine, was able to block FcR-mediated phagocytosis in THP-1 monocytic cells (Fig. 1B)
and in peripheral blood monocytes (Fig. 1C)
. These results indicated that, in contrast to PMN, monocytes present FcR-mediated phagocytosis independently of PI 3-K and ERK. The data also show that PKC is an important regulator of phagocytosis in monocytes. Although FcR-mediated phagocytosis seems to be uncoupled to PI 3-K and ERK, both enzymes are completely functional in these cells and are activated after FcR engagement (Fig. 2
). As we have shown before, activation of PI 3-K and ERK enzymes is required to activate the transcription factor NF-B, which in turn is needed for FcR-mediated induction of interleukin-1 production [4
]. FcR-mediated NF-B activation was inhibited efficiently by wortmannin and PD98059 (Fig. 3
). Together, these results suggested that although FcR cross-linking activates ERK and PI 3-K efficiently, which are needed for functions such as genen induction, FcR-mediated phagocytosis proceeds independently of these signaling molecules in monocytes.
|
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. This treatment resulted in THP-1-differentiated macrophages that grew, contrary to undifferentiated cells, attached to the tissue-culture flask (not shown). Several cell surface markers were analyzed by flow cytometry to confirm the macrophage phenotype of these cells. Surface expression of CD14 (Fig. 4A
), ß2 integrins (Fig. 4B)
, and MHC class I molecules (Fig. 4C)
clearly augmented on monocyte-differentiated macrophages. Also, surface expression of FcRI and -III (Fig. 4D
and 4F)
increased on the cell membrane of monocyte-differentiated macrophages. Higher surface expression of these molecules is typical of macrophages [25
, 26
]. In contrast, surface expression of FcRII (Fig. 4E)
and of ß1 integrins (not shown) remained unchanged. Additionally, the level of peroxidase, which is known to be much smaller in macrophages than in monocytes and neutrophils [26
], was determined in monocyte-differentiated macrophages. THP-1 cells had levels of peroxidase activity about seven times higher (Abs=0.21±0.08; mean±SD, n=3) than those of monocyte-differentiated macrophages (Abs=0.03±0.02; mean±SD, n=3). Taken together, these data indicate that our THP-1 cells had indeed fully differentiated into macrophages after retinoic acid and IFN- treatment.
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R-mediated phagocytosis by 24 h- and 48 h-differentiated macrophages was sensitive to staurosporine (Fig. 5)
. All these data showed that complete differentiation of monocytic cells into macrophages resulted in recruitment of PI 3-K and ERK for regulation of phagocytosis.
|
R cross-linking. ERK activity was inhibited by PD98059 in a dose-dependent manner. The inhibition pattern was the same for both cell types (Fig. 7A
). Similarly, PI 3-K activity was inhibited by LY294002 with the same pattern in both cell types (Fig. 7B)
. These data demonstrated that differentiation of monocytes into macrophages did not change the level of mRNA expression and enzymatic activities of ERK and PI 3-K.
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R-mediated phagocytosis might render the cells more efficient for this process. Upon stimulation with 100 ng/ml PMA, PMN and 48 h-differentiated macrophages presented significantly higher PIs than monocytes (Fig. 8
). These results support the idea that efficient phagocytes require ERK and PI 3-K for maximal FcR-mediated phagocytosis.
|
R signaling leading to phagocytosis. To explore the relationship of PKC PI 3-K and ERK further in FcR signaling in monocytes and macrophages, we looked at FcR-induced ERK activation in the presence of PI 3-K and PKC inhibitors. As shown previously, FcR cross-linking with IIC results in ERK activation. LY294002 reduced ERK activation significantly in PMN, THP-1 monocytes, and monocyte-differentiated macrophages (Fig. 9
). Staurosporine also inhibited ERK activation in PMN (Fig. 9A)
and monocytes (Fig. 9B)
, but not in macrophages (Fig. 9C)
. These results indicated that PKC is important for phagocytosis in all phagocytes, but its relationship with ERK changes when monocytes differentiate into macrophages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R-mediated phagocytosis by monocytic cells and by monocyte-differentiated macrophages. Phagocytosis of IgG-coated erythrocytes by unstimulated monocytes is independent of these two signaling molecules, but upon differentiation into macrophages, PI 3-K and ERK are needed for phagocytosis.
For phagocytosis, reports indicate that ERK is needed for ingestion of IgG-opsonized particles by PMN [8
, 9
], but there are also studies showing that ERK is not required for this function in monocytic cells [10
, 11
]. So it seems that ERK may be involved in phagocytosis in some cases but not in others. Similarly, PI 3-K has been shown to be an important molecule during FcR-mediated phagocytosis [13
14
15
16
]. In all these studies, neutrophils or macrophages, which are more efficient phagocytes than monocytes [6
, 27
], were used for phagocytosis studies. However, we demonstrated previously that in monocytes, FcR-mediated phagocytosis could proceed independently of PI 3-K [11
]. Thus, it was possible that less-efficient phagocytes (i.e., monocytes) are so, because in them, phagocytosis is not coupled to PI 3-K and/or ERK. Our results in the present report indicate that unstimulated phagocytosis by a professional phagocyte (PMN) is indeed dependent on PI 3-K and ERK, whereas less-efficient phagocytes (monocytes) do not use these signaling molecules during EIgG phagocytosis (Fig. 1) . However, macrophages also seem to require PI 3-K and ERK for phagocytosis [14
, 15
]. This requirement suggested to us that monocytes, upon differentiation into macrophages, switch to a type of phagocytosis that is now dependent on PI 3-K and ERK.
THP-1 cells cultured in retinoic acid [23
, 28
] and then IFN- [22
] for 24 or 48 h acquire a macrophage phenotype. Monocyte-differentiated macrophages, in contrast to undifferentiated THP-1 cells, presented a strong dependence on PI 3-K and ERK for phagocytosis (Fig. 5) . These data clearly confirmed our hypothesis that phagocytosis of monocytes changes, from independent of PI 3-K and ERK to dependent on both enzymes, upon differentiation into macrophages. These findings are also in agreement with a previous report showing that the monocytic cell line U937 presented FcR-mediated phagocytosis in a PI 3-K-dependent manner after it was differentiated to a macrophage phenotype by treatment with PMA for 72 h [13
]. In this study, however, basal phagocytosis of undifferentiated cells was not measured. A simple explanation for the switch in the dependence of FcR-mediated phagocytosis on ERK and PI 3-K is that these proteins may be expressed at different levels in monocytic cells before and after retinoic acid and IFN- treatment; however, this is not the case. The mRNA expression level for ERK and PI 3-K (Fig. 6)
and their enzymatic activities (Fig. 7) did not change after monocytes were differentiated into macrophages. It is thus possible that the signaling pathway regulating phagocytosis in macrophages is different from that of monocytes. Although the same molecules may be activated by FcR in different cell types, the relationship among them may be altered, thus leading to differential regulation of a given cell function.
Although previous studies have suggested that leukocytes exhibit differential requirements of signaling molecules by FcR depending on their state of activation or differentiation [18
, 29
, 30
], this is the first study clearly showing that a monocytic cell can change its molecular requirements for phagocytosis when it differentiates into a professional phagocyte. How these enzymes may be recruited for regulation of phagocytosis is not known. A possible mechanism may be the number of receptors expressed on the cell surface, as has been suggested for platelet-derived growth factor receptors [31
]. Because after differentiation macrophages express higher surface levels of FcR than monocytes, it may be possible that this is also a way to bring into play certain enzymes such as ERK and PI 3-K for particular FcR functions.
Another interesting finding in the present report was that the recruitment of these two signaling molecules for phagocytosis does not take place at the same time. During monocyte-to-macrophage differentiation, FcR-mediated phagocytosis became dependent on ERK 24 h before this process showed any dependence on PI 3-K. The reason for this time difference is not known, but it seems to be in agreement with previous studies indicating that although ERK is involved in delivering signals to bring phagocytosis into action [9
, 32
], PI 3-K is needed only for the final stages of the ingestion process [33
].
It is well known that professional phagocytes present a low basal level of FcR-mediated ingestion [6
, 34
], which increases only after cell stimulation. Our results show that when monocytic cells are differentiated into macrophages, they indeed become much more efficient phagocytes upon stimulation, just as PMN, the other professional phagocytes, do (Fig. 8)
.
These results suggest that ERK and PI 3-K are, at least in part, responsible for more efficient phagocytosis by professional phagocytes. This idea is also supported by our finding that after PMA stimulation, 24 h-differentiated macrophages, which only use ERK for phagocytosis, present lower levels of phagocytosis than 48 h-differentiated macrophages, whose phagocytic response depends on ERK and PI 3-K. This observation further supports the hypothesis that both enzymes are required for a maximal phagocytic response.
That PMA induces PKC activation and that staurosporine (a PKC inhibitor) blocked phagocytosis in monocytes and in macrophages indicates that PKC is an important element in the signaling pathway leading to phagocytosis. PKC activation upon FcR stimulation is well documented [10
, 27
, 35
36
37
38
]. PKC is also needed for ERK activation in PMN [9
, 39
] and in monocytes. However, data in this study and in previous studies [10
] indicate that although FcR stimulation activates PKC and ERK, the latter enzyme is not connected to phagocytosis in monocytes, although it is connected to activation of gene transcription via NF-B (Fig. 3 , and ref. [4
]). When monocytic cells were differentiated into macrophages, the relationship between PKC and ERK changed. In macrophages, FcR-induced ERK activation became independent of PKC (Fig. 9)
. Thus, it seems that in monocytes, PKC is used to activate ERK for gene transcription and also to initiate weak phagocytosis, probably via PLA2 and arachidonic acid release [40
41
42
]. In monocyte-differentiated macrophages, PKC is not used to activate ERK, but now PI 3-K, which leads to ERK activation, and ERK itself are used for phagocytosis.
In conclusion, this study shows for the first time that monocytic cells do not use ERK and PI 3-K for basal phagocytosis. However, upon differentiation into macrophages, these enzymes are added to the signal transduction pathway leading to phagocytosis. ERK is recruited first and PI 3-K second, supporting the idea that professional phagocytes require ERK and PI 3-K for efficient FcR-mediated phagocytosis.
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ACKNOWLEDGEMENTS |
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Received March 17, 2001; revised December 17, 2001; accepted January 17, 2002.
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