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Departamento de Inmunología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México; and
Departamento de Bioquímica, Facultad de Medicina, Universidad Complutense de Madrid, and
Sección de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain), CD69 (activation-inducer
molecule), and CD98 (4F2) induced by PHA. Accordingly, PTX also
interfered with early cell activation events such as the rise in
intracellular Ca2+ and the activation of the
Na+/H+ antiporter induced by PHA and phorbol
esters, respectively. Furthermore, this drug inhibited both the cell
cycle progression and cell proliferation of T cells induced through the
CD3/TCR complex. However, this drug did not show any effect on the cell
activation/proliferation induced by PMA plus ionomycin. Our results
indicate that PTX interferes efficiently with the activation and cell
adhesion of human T lymphocytes. These effects may be of relevance for
the clinical uses of this drug. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis (5, 6). In
addition, PTX seems to interfere with the adhesion of T-lymphoma cells
and LAK cells to endothelium and tumor target cells (7, 8). Cell adhesion molecules include a wide array of cell surface receptors that belong mainly to the selectin, integrin, and Ig superfamilies (9). Most of these molecules are expressed by leukocytes, having a key role in cell migration, inflammation, and cytotoxic phenomena (10, 11). ICAM-1, a widespread and cytokine-inducible adhesion receptor, and ICAM-2, a noninducible adhesion molecule constitutively expressed by various cell types, are counterreceptors for the leukocyte integrin LFA-1 (12). These adhesion molecules play an important role in the firm adhesion of leukocytes to endothelium (12). On the other hand, ICAM-3, which is constitutively expressed by resting leukocytes (13), is a third ligand for LFA-1 and is involved in the initial phases of the immune response (14).
Very late activation Ags (VLA) or ß1 integrins are cell membrane heterodimers that mediate interaction with extracellular matrix proteins, as well as some intercellular adhesion phenomena (15). These adhesion receptors, through their interactions with their ligands, act as costimulatory molecules, contributing to the activation of lymphocytes (outside-in signaling) (15, 16, 17, 18). On the other hand, ß1 integrins are able to increase their avidity for their ligands, mainly when lymphocytes are activated (inside-out signaling). The transition of ß1 integrins to an activated conformation (high avidity for their ligands) can also be induced by Mn2+ or some activating mAbs (16, 17, 18). Interestingly, the activation of ß1 integrins induces the appearance of neo-epitopes that can be detected with specific Abs such as the 15/7 and HUTS-21 mAbs (19, 20). Thus, it is feasible, both in vivo and in vitro, to detect the activation state of ß1 integrins, and to assess the role of these molecules under physiologic and pathologic conditions (21). Herein we studied the effect of PTX on the activation and the adhesiveness of human T lymphocytes. Specifically, we assessed the effect of this drug on the adhesion of T cells to endothelial ß1 and ß2 integrin ligands as well as on the activation of ß1 integrins induced by several stimuli. In addition, we studied the effect of PTX on early cell activation events (rise in intracellular Ca2+, and activation of the Na+/H+ exchanger), on the expression of activation Ags (CD25, CD69, CD98), and cell cycle progression induced through several activation pathways. We found that PTX is able to efficiently interfere with the activation and adhesion of human T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMNC were isolated from healthy donors by Ficoll-Hypaque gradient centrifugation. T lymphoblasts were obtained by removing non-T cells from PBMNC by nylon wool adherence, and stimulating with 5 µg/ml PHA (PHA-P; Sigma, St. Louis, MO) for 48 h. Then, cells were washed and cultured in RPMI 1640 (Whittaker, Walkersville, MD) containing 10% FCS and 25 U/ml IL-2 (R & D Systems, Minneapolis, MN). T lymphoblasts cultured for 2 to 4 days were typically used in all experiments. Freshly isolated T lymphocytes were used in several experiments and were obtained from PBMNC by rosetting with SRBCs.
Reagents
PTX was obtained from Hoechst AG (Wiesbaden, Germany), and was
used 10-5 to 10-3 M in all experiments.
The T3b anti-CD3, TP1/55 anti-CD69, TP1/36 anti-CD43,
TP1/6.2 anti-CD25, and the Lia1/2 anti-ß1, HP1/7
anti-4, and HP1N anti-L integrin
chains as well as the blocking TP1/40 anti-CD11a and HP1/2
anti-4 integrin chain mAbs have been previously
described (22, 23, 24, 25, 26). The Kim127 anti-ß2 (CD18) mAb was
kindly provided by Dr. M. Robinson (Celltech, Cambridge, U.K.). The
TS2/16 is an activatory anti-ß1 integrin mAb that has
been previously described (27). The FG1/8 mAb is an IgG
mouse
anti-human CD98 (4F2), and the 15/7 and HUTS-21 are mAbs specific
for activation epitopes of ß1 integrins (19, 20).
Chimeric ICAM-1-Fc, and VCAM-1-4D-Fc, consisting of the total
extracellular domains fused to IgG Fc fragment, were obtained as
described (28). Bisindolylmaleimide, caffeine, and verapamil were
obtained from Calbiochem (San Diego, CA).
Measurements of intracellular pH (pHi) and Ca2+ ([Ca2+]i)
The pHi and [Ca2+]i were determined using BCECF (Eugene, OR) and fura-2 (Eugene) fluorescent dyes, respectively, as previously described (29, 30). Briefly, 2.5 x 107 lymphocytes were loaded with fura-2-AM (2 µM) or BCECF-AM (2 µM) for 30 min at 37°C in Ringer solution supplemented with 1% FCS. Then, fluorescence emission was measured at 510 and 530 nm for fura-2 and BCECF, respectively, using a SLM-DMX-1000 spectrofluorometer (SLM Instruments, Urbana, IL). The emission of fluorescence of BCECF was sequentially excited at 450, 462, and 500 nm, whereas that of fura-2 was excited at 340, 360, and 380 nm. The baseline fluorescence values were obtained and then cells were stimulated with PMA or PHA; the change in cell fluorescence emission in response to these stimuli was followed by at least 20 min. The data obtained were processed to determine the pHi and [Ca2+]i values. To ascertain that the alkalinization effect induced by PMA was indeed due to the activation of the Na+/H+ exchanger, experiments in Na+-free Ringer solution were run in parallel. In addition, in some experiments of stimulation of the Na+/H+ antiporter with PMA, cells were pretreated with the protein kinase C (PKC) inhibitor bisindolylmaleimide. Last, to make a better evaluation of the effect of PHA on the intracellular levels of free Ca2+, in some experiments cells were pretreated with 5 mM caffeine (to deplete the Ca2+ stored in the sarcoplasmic reticulum) and 10 µM verapamil (to block calcium channels). Under such conditions, no significant effect of PHA on the [Ca2+]i was observed.
Cell adhesion assays
Cell adhesion assays were conducted as previously described (20, 24). Briefly, 96-well microtiter EIA II-Linbro plates (Costar, Cambridge, MA) were coated with recombinant chimeric ICAM-1-Fc (10 µg/ml), or VCAM-1-4D-Fc (5 µg/ml), and nonspecific binding sites were saturated with 1% HSA. Then, plates were washed three times with PBS, and 1 x 105 cells (T lymphoblasts or freshly isolated T cells) were added to each well. After centrifugation at 10 x g for 5 min, the plates were incubated at 37°C for 20 min. To quantify cell attachment, the plates were washed thrice with RPMI 1640, and cells were fixed with methanol/acetone (1:1), and stained with violet crystal 0.5%. Violet crystal was then extracted with sodium citrate 0.1 M, pH 4.2/ethanol, and absorbance at 540 nm was measured in an EL301 ELISA reader (Behringwerke, Marburg, Germany). All assays were run in duplicate, and results were expressed as percentage of bound cells. The absorbance of 1 x 105 cells, which were fixed and stained without previous washing, was considered as 100% of cell adhesion. Specificity of cell adhesion assays was corroborated using blocking mAb (TP1/40 anti-CD11a and HP1/2 anti-VLA-4) and BSA as substratum.
Cell aggregation assays
Homotypic cell aggregation assays were conducted as previously
described (22, 24). Briefly, T cells (1 x 105),
pretreated with various doses of PTX for 24 h, were incubated in
flat-bottom 96-well microtiter plates (Costar) in a final volume of 100
µl of complete RPMI 1640 medium. Then, the pro-aggregatory TP1/36
anti-CD43, Lia1/2 anti-ß1, HP1/7 anti-4,
Kim127 anti-ß2 (CD18), and HP1N
anti-L integrin chain mAbs were added at a
concentration of 5 µg/ml, and cells were allowed to settle at 37°C
for various periods of time. Cell aggregation was determined by direct
visualization of the plate with an inverted microscope and counting the
free cells of at least five randomly chosen fields. All assays were
conducted in duplicate and results were expressed as percentage of
aggregated cells, which was obtained by the following formula: percent
aggregation = 100 x (1 - [number of free
cells])/(total number of cells).
Cell cycle analysis
T cells stimulated with anti-CD3 mAb, PMA, or PMA + ionomycin for 12, 24, 36, and 48 h in the presence of various concentrations of PTX were analyzed for DNA content by flow cytometry. Briefly, 2 to 5 x 106 T cells were washed in PBS and resuspended in the hypotonic fluorochrome solution (propidium iodide in sodium citrate, plus 0.1% Triton X-100), and treated with RNase. Samples were kept at 4°C in the dark for 30 min before flow cytometric analysis. Cell cycle analysis was performed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), using the CellFIT Software (Becton Dickinson). Results were expressed as percentage of cells in G0+G1, S, and G2+M phases of cell cycle.
Cell proliferation assays
Cell proliferation assays were performed as described (26). Briefly, T lymphoblasts were stimulated with anti-CD3, or PMA + ionomycin for 36 h in the presence of various concentrations of PTX; these cells were further incubated for 12 h after the addition of 1.0 µCi of [3H]thymidine (6.7 Ci/mM; New England Nuclear, Boston, MA). Then, cells were harvested with a semiautomated device (MH-12 Cell Harvester; Brandel, Gaithersburg, MD), and the [3H]thymidine incorporated was quantitated using a liquid scintillation counter. All assays were run in triplicate and results were expressed as the arithmetic mean of cpm incorporated.
Flow cytometry analysis
T cells incubated with several stimuli (PMA, PHA, TS2/16 mAb, 2 mM Mn2+) in the absence or presence of several concentrations of PTX for 24 to 48 h were washed and incubated with hybridoma culture supernatants or purified, biotinylated HUTS-21 mAb, followed by washing and labeling with an FITC-labeled rabbit anti-mouse Ig or FITC-avidin. Linear and logarithmic immunofluorescences were obtained in each experiment and the fluorescence produced by the myeloma P3X63 supernatant was considered as background. The results were presented as percentage of positive cells and/or mean fluorescence intensity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of PTX on T cell adhesion to the ß1
and ß2 integrin ligands ICAM-1 and VCAM-1 is shown in
Figure 1
, A and B.
The baseline adhesion of T lymphoblasts to both ICAM-1 and VCAM-1 was
significantly inhibited by PTX at 10-3 or
10-4 M (p < 0.05, Mann-Whitney
U test). In addition, the adhesion of T lymphoblasts to
either ICAM-1, or VCAM-1 induced by PMA was also significantly
inhibited by PTX (p < 0.01, Mann-Whitney
U test). Interestingly, the cell adhesion to VCAM-1 induced
by the activatory TS2/16 mAb was not affected by PTX, at either
10-4 or 10-3 M; a nonsignificant effect of
PTX was also observed when cell adhesion was induced by 2 mM
Mn2+ or with the HUTS-21 mAb, which as TS2/16 is also a
pro-activatory Ab (20) (data not shown). Similar results were observed
when experiments were conducted using freshly isolated T lymphocytes
(Table I). The effect of PTX on T cell
adhesion to both ß1 and ß2 integrin ligands
was dose-dependent, with a maximal effect at 10-4 to
10-3 M (Fig. 2A). On the other hand,
time-response experiments revealed that the effect of PTX on
PMA-stimulated T cell adhesion to both VCAM-1 and ICAM-1 was
evident as early as after 6 h, with a maximal effect after
12 h (Fig. 2B).
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). In
contrast, and in agreement with the results of cell adhesion assays,
the activation of ß1 integrins induced by the activatory
TS2/16 mAb was nonsignificantly affected by PTX (Table II); similar
negative results were obtained using Mn2+ for the
activation of ß1 integrins (data not shown).
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L), with a variable effect on
the aggregation induced by the HP1/7 anti-4 mAb
(Fig. 3). In contrast, the cell
aggregation induced with the anti-CD43 mAb employed was unaffected
by PTX. As in the case of T cell adhesiveness, the effect of PTX on
cell aggregation was dose dependent (data not shown).
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Lymphocyte adhesiveness is closely related to the activation state
of these cells; thus, we studied the effect of PTX on the activation of
T cells induced by several stimuli. The effect of PTX on the expression
of the activation Ags CD25 (IL-2R-chain), CD69 (activation-inducer
molecule), and CD98 (4F2) induced by PHA at 72 h is shown in Table III.
PTX, at either
10-3 or 10-4 M, significantly inhibited
the expression of all activation markers studied. In contrast, PTX did
not have a significant effect on the control cell surface marker CD45.
Time course experiments showed that the inhibitory effect of PTX was
observed as early as at 24 h in the case of CD69 and at 48 h
for CD25 and CD98 (Fig. 4). The
inhibitory effect of PTX was evident on both the percentage of positive
cells and the level of Ag expression (mean fluorescence intensity), and
these two parameters showed a similar trend in the time course
experiments (not shown). In agreement with these results, the early
cell activation assays showed that both the rise in
[Ca2+]i and the activation of the
Na+/H+ antiporter were significantly diminished
by PTX. Thus, the intracellular alkalinization induced by PMA, as a
consequence of the activation of the Na+/H+
cell membrane exchanger, was significantly decreased when cells were
preincubated for 24 h in the presence of 10-4 M PTX
(0.086 ± 0.010 vs 0.042 ± 0.016 pH units, untreated vs
PTX-treated cells, p < 0.05; Figure 5 and Table IV
). A similar effect was observed on the
rise of [Ca2+]i induced by PHA (65.9 ±
13.6 vs 32.1 ± 7.2 nM of Ca2+ rise, untreated vs
PTX-treated cells, n = 3; Fig. 6).
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A). This
effect was evident at both 24 and 48 h of cell culture (Fig. 7A and data not shown). The blocking effect of PTX was also
observed when freshly isolated T cells were used, with a dose-dependent
inhibition of cell cycle progression (Fig. 7B).
However, this drug did not have a significant effect on the cell cycle
progression of T lymphoblasts induced by PMA plus ionomycin (Fig. 7A). These results were confirmed by
[3H]thymidine incorporation assays (Table V
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The homotypic aggregation of T cells is a complex phenomenon that seems to involve not only cell motility and intercellular adhesion events, but also intracellular signaling phenomena. Interestingly, we have found that PTX was able to inhibit the aggregation of T lymphoblasts induced through several integrin chains, but not that induced through CD43, a highly glycosylated molecule that is involved in the adhesion and activation of T cells (33). To gain insight into the effect of PTX on T cell adhesiveness, we explored the effect of this drug on the activation of ß1 integrins. For this purpose, we used mAbs that specifically detect activation epitopes on the common chain (CD29) of VLA molecules (19, 20). Such reporter mAbs react with VLA molecules that, upon conformational changes, increase their affinity for their ligands (19, 20). Interestingly, we found that PTX inhibits the activation of these adhesion receptors induced through intracellular signals, but not that induced by extracellular stimuli such as pro-activatory mAbs or Mn2+. These data indicate that PTX seems to interfere with intracellular activation signals of human T cells that result in integrin activation. In this regard, we have previously found that in vivo activation of ß1 integrins appears to have an important role in the pathogenesis of inflammation and several immune-mediated conditions (21).
To further explore the effect of PTX on the activation of T
lymphocytes, we analyzed the effect of this drug on the expression of T
cell activation Ags induced through various stimuli. Our results on the
inhibition by PTX of the expression of the activation Ags CD25, CD69,
and CD98 by T cells indicate that this drug is indeed able to interfere
with cellular activation pathways. In this regard, the involvement of
the IL-2R-chain (CD25) in the activation/proliferation of T cells
has widely been described (34). Furthermore, CD69 is able to
efficiently contribute to the activation and cell proliferation of
lymphocytes, and CD98 appears to be involved as well in cell
proliferation (35). Thus, it is very feasible that the down-regulation
of the expression of these activation Ags by PTX further contributes to
the immunomodulatory effect of this drug. In fact, we have found that
PTX is able to inhibit the cell cycle progression of T lymphocytes, as
well as the [3H]thymidine incorporation by these cells.
These effects are likely due both to the blockade of activatory
intracellular signals and, as a consequence, to the inhibition of
expression of activation Ags involved in cell proliferation. This point
is further supported by our findings on the effect of PTX on early cell
activation events such as intracellular alkalinization and rise in
[Ca2+]i induced by PKC activators and
mitogenic lectins, respectively. In this regard, it has been described
that poor activation of the Na+/H+ antiporter
is related to the defective T cell function seen in both systemic lupus
erythematosus and bone marrow transplantation patients (29, 30). In
addition, the rise in pHi is involved in different key cell
phenomena such as cell growth, differentiation, and proliferation
(36, 37).
It has been reported that induction of the
Na+/H+ antiporter is dependent on activation of
PKC (38). A similar dependence has been described for the expression of
the early cell activation Ag CD69 (39). Since we have found that PTX
interferes with both the cellular alkalinization induced with a PKC
activator and the expression of CD69, our data suggest that one of the
possible targets of the PTX effect is the PKC activation pathway.
Interestingly, the effect of PTX on T cell proliferation and cell cycle
progression was evident when cells were stimulated through CD3, but not
when a PKC activator plus a calcium ionophore was used. It is very
feasible that PTX is able to partially block some intracellular
signals, but our data indicate that this inhibitory effect can be
overcome with strong, nonphysiologic stimuli, such as PMA plus
ionomycin. It will be interesting to precisely elucidate the molecules
and activation pathways targeted by PTX. In this regard, Wang et al.
recently described that PTX is able to block the expression of the
c-Rel transcription factor, which is involved in lymphocyte activation
(40). However, in that work, no effect of PTX on CD25 expression was
found. The disagreement between these data and our results on CD25
expression may be due to the different cells employed in our study and
in that of Wang et al. (normal human lymphocytes and a murine cell
line, respectively). Furthermore, we have studied the expression of
CD25 at the protein level rather than at the mRNA level. Last, our data
are supported by an early work of Rao et al., which also reports an
inhibition of the IL-2 receptor -chain expression by PTX
(41).
It is worth mentioning that the concentrations of PTX employed by us may be achieved in vivo after administration of high doses of this drug (1, 5, 8, 42). Furthermore, PTX, which has been used for several years for the treatment of vascular disorders, is usually well tolerated, even at high doses, with no serious side effects. Thus, PTX is an immunomodulatory agent that might be an important additional therapeutic tool for the treatment of immune-mediated conditions in which T cells are involved.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francisco Sánchez-Madrid, Sección de Inmunología, Hospital de La Princesa, Diego de León 62, 28006 Madrid, Spain.
3 Abbreviations used in this paper: PTX, pentoxifylline; VLA, very late activation Ag; pHi, intracellular pH; [Ca2+]i, intracellular calcium; PKC, protein kinase C.
Received for publication September 25, 1997. Accepted for publication February 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by human peripheral blood mononuclear cells. Immunology 83:262.[Medline]
(TNF-)-induced T-lymphoma cell adhesion to endothelioma cells. J. Invest. Dermatol. 104:824.[Medline]
2ß1 and 4ß1 integrins in leukocyte intercellular adhesion induced through the common ß1 subunit. Eur. J. Immunol. 22:3111.[Medline]
production in natural killer cells. Eur. J. Immunol. 23:1859.[Medline]
4ß1 integrin. Nature 356:63.[Medline]
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