Induction of uPA release in human peripheral blood lymphocytes by [deamino-Cysl,D-Arg8]-vasopressin (dDAVP)

Yoshitaka Yamaguchi,1,* Kenichi Yamada,1,* Toshikazu Suzuki,3 Yu-Ping Wu,2,3 Kazuko Kita,3 Shunji Takahashi,3 Masaharu Ichinose,1 and Nobuo Suzuki3

1Department of Plastic and Reconstructive Surgery and 3Environmental Biochemistry, Graduate School of Medicine, Chiba University, Chiba 260-8670; and 2Division of Clinical Research, Sakura National Hospital, Sakura 285-8765, Japan

Submitted 24 March 2003 ; accepted in final form 4 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
[deamino-Cysl,D-Arg8]-vasopressin (dDAVP), known to be an arginine vasopressin (AVP) V2 receptor agonist, is an agent that increases fibrinolytic activity levels in plasma after its infusion into the human body. However, mechanisms underlying an increase and exact localization of the extrarenal dDAVP-responsive V2 receptor remain unclarified. Two AVP receptors, V1a and V2, and a related oxytocin (OT) receptor were found to be expressed in human lymphocytes. Furthermore, we found an increase of fibrinolytic activity in the medium of peripheral lymphocytes obtained from human volunteers less than 20 min after dDAVP infusion. The increased activity was also detected in the medium after incubating the lymphocytes in the presence of dDAVP in vitro, being highest at 20 min after the incubation. In accord with the increased fibrinolytic activity, the levels of urokinase-type plasminogen activator (uPA) in the medium were also increased. However, there was no significant difference of plasminogen activator inhibitor-1 (PAI-1), pro-uPA, and tissue-type plasminogen activator (tPA) concentrations in the medium between dDAVP treatment and control. When lymphocytes were preincubated with a V2 receptor antagonist [Adamantaneacetyl1,O-Et-D-Tyr2,Val4,Aminobutyryl6,Arg8,9]-vasopressin, the dDAVP-induced uPA increase was diminished. In contrast, preincubation with a V1 receptor antagonist, [{beta}-Mercapto-{beta},{beta}-cyclopentamethylenepropionyl1,O-Me-Tyr2,Arg8]-vasopressin, prior to dDAVP treatment resulted in a greater increase of the uPA concentration in the medium than with the dDAVP treatment alone. Thus it was suggested that dDAVP may induce uPA release from human lymphocytes via V2 receptor-mediated reaction, and also via cross-talk between V1 and V2 receptors.

arginine vasopressin; plasminogen activator; urokinase-type plasminogen activator; protease release


PROTEASE ACTIVITY IN HUMAN LYMPHOCYTES is an intriguing topic because of its involvement in various senescence-associated diseases, neural migration, or demyelination disorders (31–33), although all of the proteases involved in these disorders have not been well characterized. To elucidate the role of protease activity, we recently established a method to search for agents that increase the protease activity levels in lymphocytes freshly prepared from human peripheral blood (34). In this new method, fibrinolytic activity is estimated by incubating lymphocytes with 125I-labeled fibrin as a substrate in the presence of plasminogen. This cascade reaction amplifies the activity levels and therefore is useful for detecting protease activation events of stress response in the human body (34). In particular, this reaction assay in vitro will reflect the protease activation induced by drugs in vivo.

Arginine vasopressin (AVP) and oxytocin (OT) are cyclic nonapeptides whose actions are mediated by stimulation of specific G protein-coupled receptors classified into V1a (vascular), V1b (pituitary), and V2 (renal) receptors and OT receptors (16, 18). All members of the family have been cloned, and the affinity of cloned AVP and/or OT receptors for [deamino-Cys1,D-Arg8]-vasopressin (dDAVP) and other ligands is well described (19, 36). AVP directly elicits the contraction of smooth muscle preparation via V1 receptor activation. On the other hand, V2 receptors in renal tubular cells promote the reabsorption of water (4). It has also been reported that, in canine basilar artery, AVP causes an endothelium-dependent relaxation via the V1a receptor (9) and that, in experimental animals (28) or humans (6), 4-valine-8-D-arginine vasopressin or dDAVP causes a decrease in blood pressure that is not mediated by prostaglandins (13, 16). In rat aortic strips, dDAVP evokes endothelium-dependent vasorelaxation (40), not via the authentic V2 receptor but rather via the endothelial V1-like receptors, which may be functionally different from the V1 receptor in smooth muscle cells. OT is another posterior pituitary hormone whose primary action is to stimulate uterus contraction or milk ejection function via OT receptors (35). In myometrium, binding of OT to high-affinity receptors stimulates various biological responses, including inositol-triphosphate turnover and Ca2+ influx, similar to those induced by the binding of AVP to the V1a receptors. Interestingly, the uterus contains not only OT receptors but also V1a receptors of approximately fivefold higher density in nonpregnant conditions (2). OT has also been reported to enhance glomerular filtration rate and to have a natriuretic effect (3). In addition, OT has either diuretic or antidiuretic osmoregulatory effects depending on the presence or absence of vasopressin, which may be explained by the ability of OT to bind to the adenylate cyclase-stimulating V2 receptor in distal tubules and collecting ducts (5, 15). Because some of the organs or cells express different subtypes of AVP/OT receptors, the cross-talk between them may be involved in various unknown physiological events.

dDAVP is known to be an agonist for one of the three types of AVP receptors, namely V2 (16, 18). It was reported that a marked increase of fibrinolytic activity in plasma is observed when dDAVP is infused in humans (17). This increase paralleled that of immunoreactive tissue-type plasminogen activator (tPA), which is partly derived from vascular endothelial cells (12). It was also demonstrated that dDAVP induces the release of two coagulation factors, factor VIII and von Willebrand factor, from the vascular endothelium through the activation of a vasculoendothelial V2-like receptor (11, 16, 26). However, the mechanism by which dDAVP causes the increase of fibrinolytic activity in plasma remains unclarified.

Human peripheral lymphocytes possess binding sites for dDAVP (38), although the expression of the receptor for binding dDAVP has not been determined. In addition, neither the role of V2 receptor in lymphocytes nor the action of V2 receptor agonist in peripheral blood lymphocytes has been documented until now.

In the present study, we determined the expression of mRNA species of AVP/OT receptors that can bind to dDAVP in human peripheral lymphocytes. Then, we examined whether peripheral lymphocytes obtained from human subjects with dDAVP infusion have the ability to increase plasminogen-dependent fibrinolytic protease activity and whether lymphocytes release the proteases when incubated with dDAVP in vitro.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Agents. dDAVP and AVP were purchased from Ferring Pharmaceuticals (Copenhagen, Denmark). [{beta}-Mercapto-{beta},{beta}-cyclopentamethylenepropionyl1,O-Me-Tyr2,Arg8]-vasopressin, [Adamantaneacetyl1,O-Et-D-Tyr2,Val4,Aminobutyryl6,Arg8,9]-vasopressin, and fibrinogen were obtained from Sigma Chemical (St. Louis, MO). [125I]Na (100 mCi/ml, 17 mCi/mg) was purchased from New England Nuclear (Boston, MA).

Infusion study. dDAVP was diluted with saline to allow for the intravenous delivery of 0.4 µg/kg in 100 ml over 10 min (6). Two milliliters of whole blood samples were withdrawn from cubital veins of 12 volunteers (6 males and 6 females from 40 to 60 yr old, mean age 47.3), with EDTA as an anticoagulant, every 10 min after dDAVP infusion. As a control, samples were taken from 12 age-matched volunteers (6 males and 6 females) without dDAVP infusion. Informed consent was obtained from all of the volunteers, and this study was approved by the Human Research Committee of Sakura National Hospital.

Preparation of lymphocyte samples. Lymphocytes were prepared from peripheral blood of volunteers principally according to the method described previously (32). Briefly, each (2-ml) blood sample was diluted with the same volume of phosphate-buffered saline [PBS; 10 mM sodium phosphate (pH 7.4) containing 135 mM NaCl] and put on 7 ml of Ficoll. The samples were centrifuged at 240 g for 20 min at room temperature. After centrifugation, the thin white layer of the lymphocyte fraction, termed "buffy coat," was collected and mixed with a fivefold volume of PBS. Then, it was centrifuged at 240 g for 10 min. The pellet was suspended in RPMI 1640 medium and incubated in a 60-mm dish for 20 min at 37°C for platelet attachment. Flow cytometric analysis (FACS) with anti-CD2 antibodies proved that the lymphocyte samples we used in this study contained ~95% T cells (39). Contaminations by B cells and monocytes were negligible in this separation procedure by FACS that used anti-CD13, -CD14, -CD16, and -CD17 antibodies. The lymphocyte samples were diluted with RPMI 1640 medium to make solutions containing appropriate numbers of cells (104 to 106 cells/ml).

RNA isolation and RT-PCR. Total RNA was isolated from lymphocyte samples, MCF-7 human breast cancer cells, and human umbilical vein endothelial cells (HUVEC) by use of TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. MCF-7 cells and HUVEC were used as positive controls for expression of the three AVP receptors (V1a, V1b, and V2) and the OT receptor, respectively (23, 37). After treatment with deoxyribonuclease I (Invitrogen) to eliminate possible DNA contamination, the first-strand cDNA synthesis was carried out by use of SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) with 2 µg of total RNA and 0.5 µg of oligo(dT). Thereafter, a 1-µl aliquot of the first-strand cDNA was used together with 200 nM of each specific primer, PCR buffer (in mM: 10 Tris·HCl, pH 8.3, 50 KCl, and 1.5 MgCl2), and 1 unit of recombinant Taq DNA polymerase (TaKaRa, Kyoto, Japan) to a total volume of 25 µl. The PCR primer sequences for AVP/OT receptors used in this study were exercised according to a previous report by Thibonnier et al. (37), and glyceraldehyde 3-phosphate dehydrogenase was used as a positive control for each RNA preparation. The amplification was performed in a TaKaRa thermal cycler (model TP-400) with the following steps: initial denaturation at 95°C for 5 min, followed by 35 cycles: 95°C for 30 s, 56°C for 1 min, 72°C for 1 min, and an additional extension at 72°C for 5 min. The PCR products were visualized after electrophoresis in a 2.0% agarose gel with ethidium bromide staining.

In vitro incubation of lymphocytes with or without drugs. Lymphocyte samples were incubated in RPMI 1640 medium with various concentrations of dDAVP or AVP for 20 min at 37°C in vitro. After the incubation, supernatants were obtained by centrifugation of samples for 5 min at 300 g and used for further analysis.

Preincubation of lymphocyte samples (106 cells/ml) with receptor antagonists was performed for 20 min at 37°C. After the preincubation, samples were further incubated with or without dDAVP (10–8 M) for another 20 min at 37°C and then centrifuged at 300 g for 5 min. The supernatant was used for the protease assay.

Assay of fibrinolytic activity. Fibrinogen was labeled with [125I]Na by the chloramine-T method (7) and then used for preparing polystyrene tubes coated with 125I-labeled fibrin (29). 125I-labeled fibrinogen had a specific radioactivity of 1.0 mCi/mg protein.

The lymphocyte samples and RPMI 1640 medium without lymphocytes (as a control) were incubated in 125I-fibrin-coated tubes at 37°C for 20 min in the presence of plasminogen. The released radioactivity (counts/min or cpm) of 125I was counted as described previously (29). The radioactivity increased linearly during the incubation for 1 h.

Measurement of tPA, urokinase-type plasminogen activator, and plasminogen activator inhibitor-1 concentrations. The concentrations of tPA, pro-urokinase-type plasminogen activator (pro-uPA), and plasminogen activator inhibitor (PAI)-1 in the supernatant of the samples after in vitro incubation of lymphocytes were measured using the corresponding assay kits, Chromolize tPA Assay Kit, Chromolize uPA Assay Kit, and Imulyse PAI-1, respectively (all from Biopool International, Ventura, CA). The uPA assay was performed using AngioMax Human Urokinase (uPA) ELISA Kit (Angiopharm, O’Fallon, MO). The reaction was carried out at room temperature throughout the assay.

Statistical analysis. Values are presented as means ± SD. Statistical analysis was performed by Student’s t-test with StatView software (SAS Institute, Cary, NC).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression of AVP/OT receptors in human lymphocytes. In the beginning of the study, the mRNA expression of specific AVP/OT receptors in human lymphocytes from peripheral blood was determined by RT-PCR analysis (Fig. 1). Expression of V1a, V2, and OT receptors was observed in all lymphocytes of four independent donors. On the other hand, we could not detect the expression of the V1b receptor either in human lymphocytes or in MCF-7 cells (data not shown). Two amplified DNA bands were detected from lymphocytes as well as from MCF-7 cells when we analyzed the expression of V2 receptor mRNA (Fig. 1). According to a previous report (23), MCF-7 cells express an alternative form of V2 receptor containing the entire 106 bases of intron 2 in addition to a sequence for V2 receptor mRNA as well as normal forms. Therefore, it was suggested that both normal and alternative forms of V2 receptor mRNA are expressed in human lymphocytes.



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Fig. 1. Expression of arginine vasopressin (AVP) V1a and V2 and oxytocin (OT) receptors in human lymphocytes. MCF-7 cells (lane 1) and human umbilical vein endothelial cells (HUVEC, lane 2) were used as positive controls for expression of AVP/OT receptor mRNAs. Lanes 3–6, human lymphocytes from 4 independent donors. Arrow, amplified DNA corresponding to an alternative form of V2 receptor mRNA. GAPDH, positive control for each RNA preparation.

 
Effect of dDAVP infusion on fibrinolytic activity. Each lymphocyte sample was prepared up to 40 min after infusion in humans with or without dDAVP. The levels of fibrinolytic activity in the medium of incubated lymphocytes were significantly increased and reached the peak at 20 min after the infusion, followed by a decrease to the basal level (Fig. 2).



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Fig. 2. Fibrinolytic activity in the medium of incubated peripheral blood lymphocytes after [deamino-Cys1,D-Arg8]-vasopressin (dDAVP) infusion ({bullet}) and without infusion ({circ}). Values represent the average ± SD of 3 independent experiments.

 
Effect of dDAVP or AVP treatment on fibrinolytic activity in vitro. To investigate the kinds of proteases released in the medium, lymphocyte samples were incubated with dDAVP or AVP. The levels of fibrinolytic activity in the medium were highest at 10–8 M of dDAVP, and the dose-response curve appeared to be bell-shaped (Fig. 3A). However, AVP treatment did not result in the increased levels of fibrinolytic activity at any dose examined (Fig. 3B).



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Fig. 3. Fibrinolytic activity in the medium after incubation of lymphocytes with dDAVP (A) and AVP (B). Values represent the average ± SD of 3 independent experiments.

 
Effect of dDAVP on tPA, pro-uPA, and uPA concentrations. We next measured the concentrations of tPA, pro-uPA, and uPA in the medium of lymphocyte samples incubated with dDAVP (≤10–6 M). There were no significant differences in the tPA and pro-uPA concentrations between dDAVP treatment and control (Fig. 4, A and B). However, a significant increase in the levels of uPA was observed after incubating lymphocytes with dDAVP, showing a bell-shaped pattern with the highest level at 10–8 M dDAVP (Fig. 4C).



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Fig. 4. Dose effect of dDAVP on tissue-type plasminogen activator (tPA, A), pro-urokinase-type plasminogen activator (pro-uPA, B), and uPA (C) concentrations in the medium after lymphocyte incubation. Values represent the average ± SD of 3 independent experiments.

 
Effect of dDAVP on PAI-1 concentration. The uPA assay kit used in this study recognizes both PAI-1-free (active) and PAI-1-bound (inactive) uPA. To examine whether uPA was detected as an active form or as an inactive PAI-1-bound form, we measured levels of PAI-1 in the medium after incubating lymphocytes with dDAVP (10–8 M). No increase was observed in total levels of PAI-1 concentration up to 120 min after the incubation (Fig. 5). We also made sure that the level of fibrinolytic activity in the medium after dDAVP treatment was decreased to the basal level by the addition of PAI-1 (data not shown).



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Fig. 5. Plasminogen activator inhibitor (PAI)-1 concentration in the medium after incubation of lymphocytes with ({bullet}) and without ({circ}) 10–8 M dDAVP. Values represent the average ± SD of 3 independent experiments.

 
Effect of receptor antagonists on dDAVP-induced uPA increase. We then examined whether the uPA increase in the medium was due to receptor-mediated response in lymphocytes. When lymphocytes were preincubated with the 10–8 M V2 receptor antagonist [Adamantaneacetyl1,O-Et-D-Tyr2,Val4,Aminobutyryl6,Arg8,9]-vasopressin, uPA increase after incubating lymphocytes with dDAVP (10–8 M) was not detected as it had been without the antagonist (Fig. 6). We made sure that there was no change in uPA concentration after incubation only with the V2 receptor antagonist. Preincubation with a V1a receptor antagonist, [{beta}-Mercapto-{beta},{beta}-cyclopentamethylenepropionyl1,O-Me-Tyr2,Arg8]-vasopressin, resulted in more increased levels of uPA concentration than those by dDAVP treatment alone, although the antagonist by itself did not affect the increase (Fig. 7). However, the uPA increase by the combination of a V1a receptor antagonist and dDAVP was undetectable by the 10–7 M V2 receptor antagonist (Fig. 8).



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Fig. 6. Effect of the V2 receptor antagonist on dDAVP-induced uPA increase in the medium after lymphocyte incubation. Values represent the average ± SD of 3 independent experiments.

 


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Fig. 7. Effect of the V1 receptor antagonist on dDAVP-induced uPA increase in the medium after lymphocyte incubation. Values represent the average ± SD of 3 independent experiments.

 


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Fig. 8. Effect of the V2 receptor antagonist on the V1 antagonist and dDAVP combination-induced increase of uPA concentration in the medium after lymphocyte incubation. Lymphocyte samples were preincubated with the V2 receptor antagonist (10–8 M and 10–7 M) for 20 min at 37°C. After preincubation with the V2 receptor antagonist, samples were incubated with the V1 receptor antagonist (10–8 M) for 20 min and then with dDAVP (10–8 M) for another 20 min at 37°C. Values represent the average ± SD of 3 independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we observed increased levels of fibrinolytic activity in the medium after incubating human peripheral blood lymphocytes obtained from dDAVP-infused volunteers, possibly due to uPA released from lymphocytes. This uPA induction appeared to be involved in an AVP V2 receptor-mediated reaction that is expressed in human lymphocytes (Fig. 1).

The levels of fibrinolytic protease activity in the medium were the highest when lymphocytes were incubated with 10–8 M dDAVP in vitro. The dDAVP-induced protease activity and increase in the uPA concentration showed similar dose-response curves, bell-shaped (Fig. 3A and Fig. 4C). No increase was observed in the levels of PAI-1 up to 120 min after the incubation (Fig. 5), and the level of fibrinolytic activity in the medium after dDAVP treatment was decreased to the basal level by the addition of PAI-1 (data not shown). In the absence of lymphocytes, the treatment of culture medium with dDAVP did not result in an increase of uPA concentration (data not shown). These findings suggest that the increased levels of the fibrinolytic protease activity correspond at least in part to an increase in uPA release in the medium after incubation of human peripheral blood lymphocytes with dDAVP.

The mechanism by which lymphocytes release uPA after dDAVP treatment remains unclear. We determined the population of receptors in our preparation by PCR and showed that lymphocytes express AVP V1a, V2, and OT receptors (Fig. 1). Human vascular endothelial cells have been shown to express OT receptors (37), and, in LLC-PK1 renal epithelial cells, the uPA release by OT via V2 receptor reaction has been reported (8). It could be explained by the sequential activation of various AVP/OT receptors. However, we propose that the V2 receptor system makes a large contribution to this observed uPA release, because the preincubation of lymphocytes with the V2 receptor antagonist completely inhibited the dDAVP-induced uPA increase in the medium (Figs. 6and 8).

By contrast, the V1 receptor antagonist enhanced the dDAVP-induced increase in uPA (Fig. 7). Some V1 receptor antagonists demonstrate an agonist effect (21). The agonist property of the V1-receptor antagonist used in this study, [{beta}-Mercapto-{beta},{beta}-cyclopentamethylenepropionyl,O-Me-Tyr2,Arg8]-vasopressin, was reported to be involved in the activation of the phosphoinositide-signaling pathway (30). In our experiment, this V1 receptor antagonist alone showed little effect on the uPA induction, although preincubation of lymphocytes with the antagonist enhanced uPA increase by dDAVP (Fig. 7). Interestingly, in Chinese hamster ovary cells transfected with the V1a and the V2 receptor cDNAs, the V2 receptor-induced cAMP accumulation was potentiated by stimulation of the PLC pathway via the V1a receptor (10). If this antagonist acts as an agonist to the V1a receptor, the signal transduction system may lead to the stimulation of the V2 receptor, resulting in the enhancement of the uPA induction. Another possibility is that the V1 receptor in human peripheral blood lymphocytes has an inhibitory effect on the V2 receptor function. In the presence of the V1 receptor antagonist, this inhibition may have been cleared, so that the V2 receptor fully functioned to induce the uPA increase by dDAVP.

The uPA increase under the combination of a V1 receptor antagonist and dDAVP was undetectable after V2 receptor antagonist preincubation at the highest dose (10–7 M) (Fig. 8). This result may suggest that the enhanced uPA induction is also a V2 receptor-mediated reaction. Although the exact mechanisms of the enhancement and its inhibition remain unclear, the V1 receptor may be involved in the V2 receptor-mediated uPA induction.

In our study, AVP alone did not increase the levels of fibrinolytic activity at any dose examined (Fig. 3B). We also examined the effect of the V1 receptor antagonist on uPA releasing activity by AVP, because we apprehended the possibility that the combination of the V1 receptor antagonist and AVP might be able to increase the fibrinolytic activity. However, there was no difference in uPA concentration among lymphocytes treated only with AVP, those pretreated with the V1 receptor antagonist, and control lymphocytes (data not shown). From this result, we might speculate that dDAVP has its own V2-like receptor that is inhibited by the V2 receptor antagonist and that pretreatment of the V1 receptor antagonist helps dDAVP to bind its receptor more efficiently by occupying the neighboring V1 receptors on lymphocytes. We showed two amplified DNA bands in analyzing the expression of V2 receptor mRNA (Fig. 1). The expression of an alternative form of V2 receptor mRNA in human peripheral lymphocytes might be the explanation for this receptor mechanism. RT-PCR analysis also showed that OT receptors are expressed in human lymphocytes (Fig. 1). Most of the V1a receptor antagonists, including the one that we used in this study, have high affinity with OT receptors as well as with V1a receptors. Although the affinity is higher for V1a receptors, we could not completely exclude the possibility of cross-talk between V2 and OT receptors. Very recently, a highly specific OT receptor antagonist, FE 200 440 (Ferring), was developed with an affinity for human cloned OT receptors that was ~300-fold that for V1a receptors, whereas other OT receptor antagonists bind well to both receptors (22). When this newly developed antagonist is made available, we will be able to perform further experiments that should help to more fully explain the mechanism underlying uPA release through the AVP/OT receptor function in human lymphocytes.

To our knowledge, this is the first study to report that the levels of uPA increase in the medium after incubation of lymphocytes with dDAVP. uPA is an extracellular serine endoprotease with a multimodular structure; it has been critically involved in various biological activities, such as tissue remodeling and cell migration (14). The activities trigger a protease cascade, including digestion of the extracellular matrix and activation of latent growth factors, such as transforming growth factor-{beta} and pro-hepatocyte growth factor (20, 27). Although the protease cascade is intimately associated with inflammation and tissue repair, little is known regarding the impact of lymphocytes on these processes. In this regard, it is interesting to note that human peripheral blood lymphocytes can produce uPA. Under normal physiological conditions, the level of plasma AVP concentration is much lower than that of the dDAVP we treated in our study. So, in normal conditions, the release of uPA from lymphocytes may not occur in humans. However, we emphasize the potential significance of lymphocytes releasing uPA under special conditions, such as inflammation, perhaps leading to the increasing sensitivities of the receptors on the activated T cells or the elevated concentration of vasopressin in tissues. Increased plasma concentrations and hypothalamic content and release of AVP were reported in inflammatory disease-prone Lewis rats (24, 25).

uPA released from human peripheral blood lymphocytes might be ubiquitous at the sites of inflammation or tissue repair; therefore, lymphocytes may promote tissue remodeling and angiogenesis. uPA, as an element of the fibrinolytic cascade, also takes part in regulating cell-mediated immunity in cardiac allograft acceptor mice. Histological analysis revealed that accepted cardiac allografts express uPA in mononuclear cells (1). In human renal allograft transplantation, there has been no study reporting the role of uPA on graft acceptance. If a difference in the activity of uPA release from lymphocytes exists between the renal transplant patients and normal subjects, it would be useful to know the pathophysiology of graft acceptance. Such kinds of studies are currently in progress, and the results will be reported elsewhere.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported in part by a grant-in-aid from the Clinical Research for Evidence-Based Medicine and the Research on Specific Diseases, Health and Labour Sciences Research; a grant-in-aid from the Smoking Research Foundation; "Ground Research for Space Utilization" promoted by NASDA and the Japan Space Forum; the Nissan Science Foundation; the Japan Atomic Energy Research Institute by contract with the Nuclear Safety Research Association; the Nakatomi Foundation; the REIMEI Research Resources of Japan Atomic Energy Research Institute; a grant-in-aid from the Ministry of Education, Science and Culture, Japan; and Health Science Research grants from the Ministry of Health and Welfare, Japan.


    ACKNOWLEDGMENTS
 
Present address of K. Yamada: National Hospital Organization, Chiba-East hospital, Chiba 260-8712, Japan.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Suzuki, Dept. of Environmental Biochemistry, Graduate School of Medicine, Chiba Univ., Chiba 260-8670, Japan (E-mail: nobuo{at}faculty.chiba-u.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Y. Yamaguchi and K. Yamada contributed equally to this study. Back


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