* Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain; and
Departamento de Inmunología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, México
Correspondence: Dr. Francisco Sánchez-Madrid, Servicio de Inmunología, Hospital de la Princesa, Diego de León No. 62, 28006 Madrid, Spain. E-mail: fsanchez{at}hlpr.insalud.es
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. The inhibitory effect of PTX on leukocyte polarization and extravasation may contribute significantly to the anti-inflammatory and immunoregulatory activity of this drug.
Key Words: T lymphocytes • neutrophils • chemotaxis • chemokines
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(MIP-1), whereas neutrophils are attracted mainly by CXC chemokines, including interleukin (IL)-8. However, stromal cell-derived factor 1 (SDF-1) is a CXC chemokine that exerts its chemotactic effect largely on T and B lymphocytes. Some ILs also have a chemotactic effect on leukocytes, and it has been described that IL-15 induces lymphocyte migration and cell polarization [3
, 4
].
Pentoxifylline (PTX) is a nonselective phosphodiesterase inhibitor with important immunoregulatory and anti-inflammatory effects [7
, 8
]. This drug has a beneficial effect in different immune-mediated diseases in animal models and in humans [9
10
11
]. PTX inhibits the adhesion and activation of peripheral blood T lymphocytes in vitro [12
, 13
]. In addition, it has been described that PTX is able to block the synthesis of several cytokines, including tumor necrosis factor (TNF-), likely by inhibition of nuclear factor-
B (NF-B), by preventing c-Rel activation specifically [14
]. Several studies have described the effect of PTX on distinct functions of neutrophils, including chemotaxis and adherence to hydrogen peroxide-treated endothelium [15
, 16
]. In addition, the effect of PTX on EC function under different conditions has been informed [17
18
19
]. However, an overall evaluation of the effects of PTX on phenomena that are involved in the polarization and extravasation of lymphocytes and neutrophils has not been shown. Herein, we have explored the regulatory effect of this drug on the polarization, adhesion, and migration of lymphocytes and neutrophils, the main components of most inflammatory cell infiltrates. PTX exhibited a down-modulatory effect on these phenomena. These results further support the immunoregulatory properties of PTX and related compounds.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies, cytokines, and reagents
The following monoclonal antibodies (mAb) were used: anti-ß-actin (AC-15; Sigma Chemical Co.); anti-L selectin (Leu-8; Becton Dickinson, Mountain View, CA); anti-ICAM-3 (TP1/24); anti-ß2 integrin (TS1/18) [21
]; anti-Mac-1 (Bear-1) [22
]; anti-CD31 (TP1/15) [23
]; anti-CD151 (Lia1/1) [24
]; antiactivation neo-epitope in ß2 integrins (CBRM 1/5) [25
]; and anti-E-selectin (Tea2/1) [26
]. The anti-ICAM-1 (Hu 5/3) was kindly provided by Dr. F. W. Luscinskas (Harvard Medical School, Boston, MA); anti-ICAM-1 (MEM 111) was a generous gift of Dr. V. Horejsi (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska, Czech Republic); and anti-VCAM-1 (4B9) was kindly donated by Dr. J. M. Harlan (Department of Medicine, University of Washington, Seattle). P3X63 myeloma culture supernatant or the proper isotype-matched mAb was used as a negative control. Recombinant human RANTES and MCP-1 were obtained from R&D Systems (Minneapolis, MN), and SDF-1 and IL-8 were from PeproTech (London, UK). Recombinant human IL-15 was provided by Immunex Corp. (Seattle, WA), and C5a and formyl-Met-Leu-Phe (fMLP) were purchased from Sigma Chemical Co. PTX was obtained from Hoechst AG (Wiesbaden, Germany).
Cell polarization assays
Lymphocyte and neutrophil polarization assays were performed as described [4
, 27
]. Briefly, 2 x 106 T cells or T lymphoblasts, pretreated or not with different concentrations of PTX, were allowed to adhere to coverslips precoated with fibronectin (FN) for 1 h at 37°C, 5% CO2 in 24-well plates (Costar, Cambridge, MA). Then, cells were stimulated or not with RANTES (10 ng/ml), SDF-1 (100 ng/ml), MCP-1 (10 ng/ml), or IL-15 (10 ng/ml) for 30 min and fixed. Finally, cells were immunostained for ICAM-3, and the proportion of uropod-bearing cells was calculated using a Nikon Labophot-2 epifluorescence microscope. Where indicated, the leading edge of peripheral blood lymphocytes was stained for ß-actin. In additional experiments, T lymphoblasts or Peer Aa10 cells, treated or not with PTX, were allowed to adhere to FN-coated coverslips. Then, coverslips were mounted on Attofluor open chambers (Molecular Probes, Eugene, OR) and placed on the microscope stage. Cells were maintained at 37°C in a 5% CO2 atmosphere using an incubation system (La-Con GBr, Germany), and confocal images were obtained using a Leica TCS-SP confocal laser unit. Neutrophil polarization assays were performed as follows: HUVEC were grown to confluence on gelatin-coated coverslips and activated or not with TNF- (20 ng/ml; R&D Systems). Then, purified neutrophils (1x106) were allowed to adhere to the EC for 5–15 min at 37°C, followed by fixation with 3% paraformaldehyde. In other experiments, neutrophil polarization was induced with IL-8 or C5a and performed on coverslips precoated with FN80. The immunostaining for ICAM-3 and calculation of the proportion of polarized cells were made as described for lymphocytes.
Chemotaxis and transendothelial-migration assays
T cells or T lymphoblasts (1x106), pretreated or not overnight with different doses of PTX, were added to the upper compartment of Transwell cell-culture chambers, 5 µm-diameter pore size (Costar), and 600 µl of the indicated chemokine was poured to the lower well. Then, cells were incubated for 90 min at 37°C in 5% CO2, and migrated cells were recovered from the lower chamber and counted during 60 s using a FACscan flow cytometer (Becton Dickinson). In transendothelial migration assays, HUVEC were grown to confluence on Transwell inserts precoated with gelatin and activated or not with TNF- (20 ng/ml) for 6 h. T lymphocytes (1x106), treated or not overnight with PTX, were added to the upper chamber, and 600 µl of the indicated chemokines were added to the lower compartment. Cells were incubated for 16 h at 37°C, and migrated cells were counted as indicated. Neutrophil chemotaxis assays were performed during 30 min using, when indicated, the following chemoattractants: IL-8 (2.5 nM), C5a (0.5 nM), or fMLP (1.0 nM). The number of migrated cells was also estimated by flow cytometry.
Neutrophil-HUVEC adhesion assay
HUVEC grown to confluence on gelatin-coated coverslips in 24-well plates were activated or not overnight with TNF- (20 ng/ml). Neutrophils (1x106 in 500 µl M199 medium with 0.05% human serum), preloaded with the fluorescent probe BCECF-AM (Molecular Probes), were treated or not with the indicated doses of PTX for 30 min and allowed to adhere to the EC monolayer for 10 min at 37°C, 5% CO2. Then, coverslips were washed, fixed, and visualized in a Nikon Labophot-2 microscope. The number of neutrophils (fluorescent cells) that adhered to endothelium was counted in 10 random fields, and results were expressed as the total number of cells registered.
Flow cytometry analysis
Neutrophils (1x106 cells/ml) were incubated in the presence or not of the indicated doses of PTX for 30 min and stimulated with IL-8, C5a, or fMLP for 15 min at 37°C. Then, cells were stained for L-selectin, Mac-1, and ICAM-3 using specific mAb plus a fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse immunoglobulin G (IgG; Dako, Carpinteria, CA). Cells were analyzed by flow cytometry, and results were expressed as the percent of positive cells or the mean fluorescence intensity (MFI). The effect of PTX on endothelium was assessed using confluent HUVEC cultures pretreated or not with the indicated doses of PTX for 8 h and stimulated or not with TNF- (20 ng/ml) overnight. Then, cells were detached with trypsin-ethylenediaminetetraacete (EDTA), washed immediately, and stained for the expression of E-selectin, VCAM-1, and ICAM-1 with specific mAb, followed by an FITC-labeled rabbit anti-mouse IgG (Dako). Cells were analyzed by flow cytometry.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and cytokines, such as IL-15 (Fig. 1A
). The effect of PTX was dose-dependent, with a significant inhibition at 10-4 M. Kinetics analysis showed a maximal inhibitory effect at 12–18 h after PTX pretreatment of the cells (unpublished results). In separate experiments, we corroborated that PTX inhibited the relocation of ICAM-3 to the uropod and the concentration of ß-actin at the leading edge induced by SDF-1 (Fig. 1B)
. PTX also inhibited the polarization of cultured T lymphoblasts induced by IL-15, RANTES, or MCP-1 (Fig. 2A
and 2B
). In contrast, PTX had no significant effect on the polarization of Peer Aa10 lymphoid cells expressing ICAM-3/enhanced green fluorescence protein (Fig. 2C)
. The polarized cluster of ICAM-3 in these cells remained unaffected after treatment with 10-3 M PTX for 48 h (Fig. 2C)
, suggesting that PTX inhibits the induction but not the maintenance of a polarized structure.
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Figure 1. Effect of PTX on the polarization of PBMC. (A) PBMC, pretreated or not with the indicated doses of PTX, were allowed to adhere to FN80-coated coverslips and were stimulated with IL-15 or SDF-1. Then, cell polarization was assessed by immunostaining for ICAM-3, as described in Materials and Methods. Data correspond to the arithmetic mean ± SD of the percent of polarized cells from five independent experiments. (B) Double-immunostaining for ß-actin (green) and ICAM-3 (red) in PBMC pretreated or not (-) with PTX (10-4 M) and stimulated or not with SDF-1 (100 ng/ml). Images from a representative experiment are shown.
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Figure 2. Effect of PTX on the polarization of T lymphoblasts. T lymphoblasts, pretreated or not with the indicated doses of PTX and adhered to FN80-coated coverslips, were stimulated with the indicated cytokines. Then, cell polarization was assessed as stated in Materials and Methods. (A) Images of a representative experiment. (B) Quantification of the effect of PTX on cell polarization. Data correspond to the arithmetic mean ± SD of the percent of polarized cells from five independent experiments. (C) Peer Aa10 lymphoid T cells were incubated in the presence of 10-3 M PTX and observed by live confocal microscopy for 48 h as stated in Materials and Methods. Images correspond to 15 h of incubation. No effect on cell shape and motility was observed.
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. A similar effect on the chemotaxis of cultured T lymphoblasts induced by IL-15 was observed (Fig. 3B)
. This effect was seen as early as after 45 min of incubation with PTX, with a maximal inhibition at 12–18 h after PTX treatment (unpublished results). The random migration of lymphocytes (Fig. 3A and 3B
, Medium) was also inhibited significantly by PTX. In addition, the basal transendothelial migration of lymphocytes as well as that induced by SDF-1 was also inhibited significantly by this drug (Fig. 3C)
. However, when transendothelial migration assays were performed on activated endothelium and in the presence of optimal amounts of SDF-1, no inhibitory effect of PTX was seen (Fig. 3C)
. Therefore, PTX interferes with the basal migration and its response to different stimuli, but the activation of endothelium overcomes this effect partially.
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Figure 3. Effect of PTX on the migration of lymphoid cells induced by cytokines. PBMC (A) or cultured T lymphoblasts (B), pretreated or not with PTX, were poured in the upper compartment of Transwell chemotaxis chambers. Then, cell migration was stimulated with IL-15 and/or SDF-1 and quantified as described in Materials and Methods. (C) The upper side of the Transwell membrane was coated with a confluent endothelial cell monolayer and pretreated or not with 20 ng/ml TNF- for 4 h. Data at the left and right of the dotted line correspond to their respective scales. Data correspond to the arithmetic mean ± SD of the number of migrated cells from eight (A, B) and five (C) independent experiments. Asterisks indicate significant differences (P<0.05, Mann-Whitney U-test) compared with untreated cells.
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for 4 h resulted in an increase in neutrophil adhesion and a significant decline of the inhibitory effect of PTX.
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Figure 4. Effect of PTX on neutrophil adhesion and migration. (A) BCECF-labeled neutrophils, pretreated or not with PTX 10-3 M for 30 min, were stimulated or not with IL-8 (2.5 nM, 15 min) and allowed to adhere to resting or activated (TNF-, 20 ng/ml, 4 h) confluent, endothelial cells for 15 min at 37°C. Then, cell adhesion was quantified by fluorescence microscopy, as described in Materials and Methods. When indicated (mAb), a mixture of blocking anti-E-selectin (Tea2/1), anti-ß2-integrins (TS1/18), and anti-ICAM-1 (Hu-53) mAb were added to the assay. Data from a representative experiment out of three are shown. (B) Nonstimulated neutrophils were pretreated or not with PTX and poured on the top compartment of a Transwell chemotaxis chamber with a polycarbonate membrane precoated with confluent endothelial cells. Then, cell migration was allowed for 30 min, and the number of migrated cells was estimated by flow cytometry, as described in Materials and Methods. When indicated, purified anti-CD31 (20 µg/ml) or anti-CD151 (20 µg/ml) mAb were added to the upper chamber 15 min before addition of neutrophils. (C) In similar assays, neutrophil migration was induced, when indicated, with IL-8 (2.5 nM), C5a (0.5 nM), or fMLP (1.0 nM). Data correspond to the arithmetic mean ± SD from five independent experiments. Asterisks indicate significant differences (P<0.05, Kruskal-Wallis and Mann-Whitney U tests).
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, 20 ng/ml, for 18 h), no significant effect of PTX was observed (unpublished results). As expected, PTX-treated neutrophils exhibited a diminished ability to spread and polarize on activated EC (Fig. 6A
). PTX also had a clear-cut blocking effect on the induction of neutrophil polarization when these cells were attached to FN80 and stimulated with IL-8 or C5a (Fig. 6B
; and unpublished results).
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Figure 5. Kinetics and dose-response analyses of the effect of PTX on neutrophil transmigration. Nonstimulated human neutrophils were pretreated with the indicated doses of PTX for 30 min (A) or with PTX 10-3 M for the indicated times (B) and assayed for transendothelial migration on Transwell chemotaxis chambers, as described in Materials and Methods. Data correspond to the arithmetic mean ± SE of three independent experiments.
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Figure 6. Effect of PTX on neutrophil polarization. (A) Nonstimulated neutrophils, pretreated or not with PTX 10-3 M for 30 min, were allowed to attach to activated endothelium (TNF-, 20 ng/ml, 24 h) for 15 min. Then, cells were stained for ICAM-3. Images correspond to a representative experiment out of four performed. (B) Neutrophils (2x105), pretreated or not with PTX 10-3 M, were stimulated or not with IL-8 (2.5 nM) and allowed to adhere and polarize onto FN-80-coated coverslips for 15 min. Then, cells were fixed, immunostained for ICAM-3, and analyzed by immunofluorescence microscopy. Results of a representative experiment out of five performed are shown. Numbers correspond to the percent of inhibition of cell polarization with respect to PTX-untreated cells. Microphotographs of neutrophils treated with IL-8 alone and IL-8 plus PTX are also shown.
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-induced expression of E-selectin and VCAM-1 by HUVEC (Fig. 7
). However, this drug had an evident down-regulatory effect on the enhancement of expression of ICAM-1 during EC activation. This effect was observed when EC were incubated in the presence of TNF- for 4 h but not after 16 h of incubation (unpublished results). |
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Table 1. Effect of PTX on the Expression of Adhesion Molecules by Human Neutrophils
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Figure 7. Inhibitory effect of PTX on ICAM-1 expression by endothelial cells. HUVEC were pretreated or not with PTX 10-3 M for 16 h and then activated with TNF- (20 ng/ml) for 4 h. Expression of activation molecules was analyzed by flow cytometry, as described in Materials and Methods. Dotted line, nonactivated cells; thin line, activated cells with no PTX treatment; thick line, activated cells pretreated with PTX. Data from a representative experiment out of four are shown.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The recruitment of T cells into inflammatory cell infiltrates requires their activation, adhesion, polarization, and locomotion. Previously, we have found that PTX exerts a down-modulatory effect on the activation and adhesion of T lymphocytes [13 ]. Accordingly, herein, we demonstrate that this drug is able to interfere with polarization and chemotaxis of these cells. Lymphocyte polarization involves important changes in cell shape, with the formation of a leading edge and a cytoplasmic projection at the rear of the cell—the uropod [3 , 31 ]. Different cell-adhesion molecules, including ICAM-1, ICAM-3, PSGL-1, and CD44, are redistributed to the uropod during cell polarization, with the involvement of cytoskeleton and linker proteins [3 ]. Lymphocyte polarization is induced by different cytokines, mainly IL-15 and chemokines. However, the intracellular signals involved in cell polarization have been disclosed only partially. Previously, we have described the participation of PI-3K, PKA, and Rho GTPases in the induction of cell polarization [32 , 33 ]. In this regard, it is feasible that PTX, through its inhibitory activity on phosphodiesterases and the sustained rise in cAMP, interferes with the intracellular mechanisms responsible for cell polarization. It is interesting that PTX did not exert any inhibitory effect on the constitutively polarized Peer cells, suggesting that there are some signaling pathways or biochemical steps involved in the activation and/or the maintenance of polarization in lymphoid cells that are not affected by PTX. This point is supported by the lack of an inhibitory effect of PTX on the activation of T cells induced by PMA/ionomycin but with a clear-cut effect of this drug on the stimulation through the TcR/CD3 complex [13 ]. Further studies are necessary to elucidate the precise inhibitory mechanism of PTX on cell polarization.
Cell polarization is closely related to cell motility and leukocyte recruitment to inflammatory foci [1 , 3 , 31 ]. Thus, the inhibitory effect of PTX on cell polarization is in agreement with its down-regulatory activity on lymphocyte chemotaxis. It is interesting that PTX interfered with the chemotaxis induced through two different types of receptors, those for chemokines and those for IL-15. Although the intracellular signals induced through these receptors seem to be different, it is clear that PTX is able to interfere with a common signaling pathway involved in cell polarization/chemotaxis and that this effect may be related to the anti-inflammatory activity of PTX observed in different experimental models [7 8 9 ]. Furthermore, the effect of PTX on lymphocyte polarization may have important functional consequences in addition to inhibition of chemotaxis. It has been described that lymphocyte polarization is involved in the immune interactions necessary for the generation of the immune response [34 35 36 ]. In addition, T lymphocytes interact with antigen-presenting cells through their leading edge, where an enhanced sensitivity to antigen is found [34 ]. Furthermore, it has been described that the cytotoxic activity of T and natural killer cells requires the maintenance of a polarized shape [37 , 38 ]. Finally, additional, novel functions are emerging for the accumulation of molecules to the uropod, including the regulation of T-cell apoptosis by redistribution of CD95 [39 ] and the sequestration of growth factors by the concentration of proteoglycans [40 ]. Therefore, PTX interferes with cell phenomena that have a pivotal role in the induction and effector phase of the immune response. However, our results with the Peer Aa10 cells indicate that not all lymphoid cells are sensitive to the effect of PTX.
Neutrophils constitute the other leukocyte population that is a key element of inflammatory phenomena. In this case, polymorphonuclear leukocytes are mainly recruited to acute inflammatory cell infiltrates. In agreement to its effect on lymphocytes, PTX also inhibits the polarization and migration of neutrophils. In addition, the interaction of these cells with endothelium is down-regulated by PTX, including intercellular adhesion, cell spreading, and transmigration. Furthermore, PTX inhibits neutrophil activation, as revealed by its effect on the expression of Mac-1 and its activation epitope CBRM 1/5 [25
]. The process of neutrophil polarization is similar to that observed in lymphocytes, with concentration of different cell-adhesion molecules at the uropod, including ICAM-3 and PSGL-1 [27
]. The concentration of adhesion molecules at the uropod facilitates the interaction of the polarized leukocytes with other cells, allowing their recruitment into the inflammatory cell infiltrate [3
, 41
, 42
]. Thus, the inhibition of neutrophil polarization/activation by PTX accounts, at least in part, for the anti-inflammatory effect of this drug. In this regard, it has been shown that PTX has a beneficial effect in conditions in which neutrophils have a key role, such as the septic shock and reperfusion damage [43
, 44
]. All these data suggest that PTX may exert an important antiinflammatory activity. However, our results indicate that the inhibitory effect of PTX may be overcome by potent physiological stimuli. Thus, this drug had no effect on the transmigration of neutrophils across a fully activated EC monolayer nor in the transmigration of lymphocytes induced by SDF-1 across an activated EC monolayer. It is possible that a similar phenomenon may occur in vivo under different clinical conditions. Finally, it is interesting that in contrast with nonsteroidal, anti-inflammatory drugs [45
], PTX does not modify the expression of L-selectin. These data are in agreement with the lack of correlation between intracellular levels of cAMP and shedding of L-selectin [46
] and make evident the different mechanisms of PTX action and other antiinflammatory drugs.
Activated endothelial cells express different adhesion molecules that allow their interaction with bloodstream leukocytes. ICAM-1, VCAM-1, and P- and E-selectins mediate the rolling and firm adhesion of leukocytes on activated endothelium [1
, 2
]. It is interesting that our data indicate that PTX has a selective, down-regulatory effect on ICAM-1 expression during the activation of HUVEC by TNF-. It is conceivable that this effect could also contribute to the anti-inflammatory effect of PTX. However, it remains an interesting question as to how PTX exerts a selective effect on the expression of a single cell-adhesion molecule by EC.
In summary, our data indicate that PTX exerts an important down-regulatory effect on the main cellular elements of chronic and acute inflammatory cell infiltrates, with a mild but significant inhibitory activity on the other key component of these phenomena, the EC. Remarkably, although most of the inhibitory effects of PTX described in the present work were more evident at high concentrations (
10-4 M), it is worth mentioning that high plasma levels of PTX can be achieved after the intravenous administration of this drug [47
]. In addition, in some conditions (e.g., liver cirrhosis), very high plasma levels of PTX are observed without apparent toxicity [47
]. Thus, we think that it is likely that the inhibitory effects of PTX observed by us in vitro may also occur in vivo, mainly after the prolonged administration of high doses of this drug (e.g., 400 mg, three in a day). However, our data also indicate that when strong proactivatory stimuli are used in vitro, some of the inhibitory effects of PTX are lost. Therefore, our data indicate that although PTX is a very interesting drug with relevant immunoregulatory and anti-inflammatory activities, it is important to search for pharmacological agents with similar properties and effects but with a higher potency.
Received October 30, 2001; revised October 30, 2001; accepted December 10, 2001.
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3ß1 integrin localized at endothelial lateral junctions J. Cell Biol. 141,791-804 by human peripheral blood mononuclear cells Immunology 83,262-267[Medline]-induced lymphocyte polarization and chemotaxis J. Immunol. 163,4001-4012This article has been cited by other articles:
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