Transmembrane signalling in human monocyte/mesangial cell co-cultures: role of cytosolic Ca2+
Paolo Menè,
Francescaromana Festuccia,
Rosaria Polci,
Francesco Pugliese and
Giulio A. Cinotti
Division of Nephrology, University La Sapienza of Rome, Italy
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Abstract
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Background. Adhesion of monocytes triggers apoptosis, cytotoxicity, cytokine release, and later proliferation of cultured human mesangial cells (HMC). In the search for transmembrane signals transducing the interaction of HMC adhesion molecules with leukocyte counterreceptors, we measured variations of cytosolic Ca2+ ([Ca2+]i) in HMC and monocytes of the U937 cell line during 6-h co-cultures.
Methods. Monolayer cultures of HMC and suspensions of U937 cells were loaded with the fluoroprobe fura 2-AM and subsequently co-cultured for 6 h while separately monitoring by microfluorometry the Ca2+-dependent 500 nm fluorescent emission of each cell line at fixed intervals upon excitation at 340/380 nm.
Results. U937 and peripheral blood monocyte adhesion was followed in HMC by a slow, progressive rise of [Ca2+]i from basal levels of 96±9 nM to 339±54 at 60 min and 439±44 nM at 3 h. The [Ca2+]i elevation reached a steady state thereafter, while parallel monolayers incubated with control media maintained resting levels throughout the co-culture with stable fluoroprobe retention. Receptor sensitivity to vasoconstrictor agents, including compounds not released by monocytes, such as angiotensin II, was rapidly downregulated in HMC co-cultured with U937 cells. No [Ca2+]i changes could be elicited by the octapeptide or by the TxA2 analogue, U-46619, as early as 30 min after exposure to U937 cells. No [Ca2+]i changes were observed in U937 cells throughout the co-culture. Conditioned media from monocytes and from co-cultured HMC+U937 cells had no effect on [Ca2+]i of HMC. Ca2+ entry leading to fura 2 saturation was still inducible by Ca2+ ionophores, such as ionomycin and 4-Br-A23187, which also inhibited the responses to vasoconstrictors. Ca2+-free solutions prevented the [Ca2+]i rise as well as subsequent receptor inactivation, implicating Ca2+ influx through store-operated Ca2+ channels (SOC), a major pathway for Ca2+ entry in these cultured cells. Ca2+ influx was confirmed by Mn2+-quenching of fura 2.
Conclusions. In HMC, early changes in [Ca2+]i signal for monocyte adhesion in a co-culture model of glomerular inflammation. This signalling mechanism may mediate the functional responses elicited in glomerular cells by leukocytes, including downregulation of receptors for vasoactive agents.
Keywords: Ca2+ channels; cytosolic free Ca2+; mesangium; monocytes; signalling
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Introduction
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Several forms of glomerulonephritis, vasculitides, interstitial nephritides, ureteral obstruction and renal allograft rejection are typically associated with leukocyte chemoattraction and accumulation within the kidney [13]. The cross-talk between blood-borne cells and renal cells involves both diffusible mediators, such as cytokines, chemokines and growth factors, as well as the direct interaction of membrane-bound integrins and adhesion molecules belonging to the immunoglobulin superfamily [4,5]. Leukocyte products including cytokines, enzymes, and bioactive lipids can be recovered in renal biopsies or in the urine as markers of kidney infiltration and inflammatory reactions [6,7]. Co-culture of leukocytes and kidney cells has been extensively employed in recent years to investigate the cellular background of inflammation. We have developed an assay based on the adhesion of undifferentiated monocytoid cells of the U937 line, ordinarily growing in suspension, to monolayers of cultured human mesangial cells (HMC) through a mechanism involving matrix components, adhesion molecules and their respective leukocyte counterreceptors [8,9]. This process results in functional changes of HMC, which undergo cytotoxic cell damage when exposed to viable U937 cells or are primed to release cytokines upon internalization of apoptotic monocytes, as shown by other investigators [10,11]. A later proliferative response ensues, which requires persistent contact between monocytes and HMC, and cannot be induced by U937-conditioned media [9]. Diffusible mediators are therefore unlikely to play a role in this model.
In the present investigation, we asked what are the immediate signalling mechanisms leading to HMC responses to peripheral blood or U937 monocytes. In view of the extensive literature on the cytosolic free Ca2+ ([Ca2+]i) changes occurring upon activation of cytotoxic lymphocytes (natural killer cells) or macrophages [12,13], we hypothesized that early changes of [Ca2+]i could be employed by either cell population to signal for docking of a monocyte and the resulting HMC response. We then undertook a monitoring of [Ca2+]i in both cell lines during 6-h co-cultures, screening the mechanisms by which Ca2+ fluxes could be altered by this cellular interaction.
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Methods
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Cell culture
Pure lines of HMC were obtained with standard techniques from glomerular explants [14,15]. Kidneys not suitable for transplantation or nephrectomy specimens histologically free of lesions were used after obtaining the written informed consent of patients or relatives. Four independent cell lines were used in passages 318. RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Flow Laboratories, Irvine, UK), 5 µg/ml human recombinant insulin (Novo, Copenhagen, Denmark), 10 µg/ml ceftriaxone (Hoffmann-La Roche, Basel, Switzerland) or 100 µg/ml gentamycin (Fournier Pierrel, Milan, Italy) was used for initial plating and propagation of the cultures. The cells were maintained at 37°C in a controlled, humidified atmosphere of 95% O2-5% CO2 and subcultured every 47 days in complete RPMI medium whenever approaching confluence.
Undifferentiated U937 cells (ATCC code CRL 1593) were grown in suspension cultures employing the same FBS-supplemented RPMI 1640 medium. Cultures were expanded by seeding 2x106 cells into 25 ml in 75 cm2 flasks (Costar, Cambridge, MA, USA) every 72 h. Fresh medium was added every 24 h. Prior to an experiment, the suspension was centrifuged at 1000 g for 10 min, the cells rinsed once in serum-free RPMI 1640, counted and spun again before final resuspension at the density of 1x106 cells/ml. In selected experiments, U937 cells were layered onto MillicellTM-HA culture plate inserts (Millipore, Bedford, MA, USA) placed into each well of a 12-well dish containing the HMC coverslips, to ensure diffusion of the bathing medium between both monocytic cells and HMC, without actual direct physical interaction.
Preparation of human peripheral blood monocytes (PBM)
PBM were isolated by selective adhesion on plastic from a suspension of lymphomonocytes obtained by centrifugation of whole blood (30 ml) diluted 1:1 v/v with normal saline on LymphoprepTM (Nycomed Pharma, Oslo, Norway; dilution 2:1 v/v). After washing in phosphate-buffered saline (PBS, pH 7.2), the mononuclear cell pellet (>95% purity, free of platelets by phase-contrast microscopy after WBC lysis) was resuspended in RPMI 1640 medium supplemented with 0.5% FBS and plated onto 6-well culture dishes. After 60 min incubation at 37°C, non-adherent lymphocytes were dislodged by 3x washing with fresh medium. The adherent monocytes in each well were then resuspended by incubation for 15 min in Ca2+-free PBS, followed by gentle scraping with a rubber policeman, and counted by haemocytometry prior to dilution at the desired density for the adhesion assays.
[Ca2+]i measurements
[Ca2+]i was measured fluorometrically in cells loaded with the intracellular probe, fura 2 (Molecular Probes, Eugene, OR, USA) as previously described [1416]. Briefly, after withdrawing FBS for the 24 h prior to an experiment, confluent HMC monolayers grown on plastic Aclar coverslips (Allied Engineered Plastics, Pottsville, PA, USA) were selectively loaded for 40 min at 37°C with 1 µM fura 2 in serum-free RPMI 1640, followed by further incubation for 20 min in the same medium without fura 2. Fluorescence measurements were performed by inserting the coverslips diagonally in a quartz cuvette filled with 2 ml of modified Krebs-Henseleit solution (KHH) of the appropriate glucose concentration, buffered with 20 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) and supplemented with 0.2% fatty acid-free bovine serum albumin. Whenever appropriate, a suspension of non-labelled U937 cells was injected into the cuvette to a final density of 1x106 cells/ml. The monolayers were excited at 340 nm with emission collected at 500 nm in a Perkin-Elmer LS5B spectrofluorometer and thermostatically controlled cell. Continuous gentle stirring of the suspension was maintained by a remotely controlled Rank mod. 200 electronic stirring apparatus. Excitation/emission slits were set at 2.5/5 nm, respectively. Alternatively, U937 cells were separately loaded with fura 2 by resuspension in serum-free RPMI 1640 containing 1 µM of the fluoroprobe. After 40 min the cells were spun and allowed to recover in KHH solution prior to centrifugation and resuspension in the experimental cuvette. Calibration of Ca2+-dependent fluorescence was performed by sequential saturation of the dye with 15 to 40 µM ionomycin (Calbiochem-Behring, La Jolla, CA) ±10 mM CaCl2 (HMC), or by permeabilizing U937 cells with 50 µM digitonin to maximum fluorescence (Fmax), followed by chelation of Ca2+ to minimum fluorescence (Fmin) with 7.5 mM ethylene glycol-bis (ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) plus 60 mM Tris, pH 10.5. Ratio fluorometry with alternate 340/380 nm excitation at 6 s intervals was employed for validation of each set of experiments. Standard formulae were employed for the calculation of [Ca2+]i, employing a Kd of fura 2 for Ca2+ of 224 nM [16]. The autofluorescence of the monocyte suspension was subtracted when monitoring [Ca2+]i in HMC. During prolonged co-culture, to ensure substantial fura 2 retention and prevent photobleaching, fluorescent light entry into the cuvette was blocked by an excitation shutter, which was released only for 35 min recordings of fluorescence at each experimental interval. In the Mn2+-quenching experiments, the fluorescence decay of fura 2 was monitored with excitation set at 340 nm and at the 360 nm isosbestic point for fura 2 for at least 3 min after addition of 100 µM MnCl2, and then for additional 3 min after application of the vasoconstrictors employed to promote Ca2+ influx [17].
Statistical analysis
All data were expressed as mean±SE and analysed by one-way analysis of variance.
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Results
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Earlier work from our and other laboratories established that monocytoid cells of the U937 line adhere to cultured HMC in a specific and irreversible manner. As these cells ordinarily grow and proliferate in suspension, their firm adhesion to HMC and/or extracellular matrix is mediated by surface integrins recognized by leukocyte counterreceptors [4,5,8,9]. The present studies confirmed and extended these findings, showing that a slow rise of [Ca2+]i occurs in HMC exposed to U937 cells for up to 6 h. Figure 1
shows the pattern of such response when [Ca2+]i was monitored at fixed intervals after selective loading of HMC with fura 2. Under these conditions, the intracellular tracer was substantially retained, as confirmed by the stable Fmax/Fmin ratiometric readings upon calibration of a monolayer at the end of the 6-h observation or when individual calibration was obtained in independent monolayers for each experimental point (not shown). No photobleaching of fura 2 occurred, as the 340/380 nm excitation was limited by a shutter to 3 min intervals at each experimental point. The constant presence of freely floating, unbound U937 cells was ensured by continuous slow stirring of the bathing medium, so that any diffusible mediator generated during the incubations with excess monocytes was not lost. At the same time, an average binding of 0.9±0.09 U937 cells per HMC was confirmed by repeated fixation and staining by the May-Grünwald-Giemsa method (n=12) of the monolayers after 3 h of co-culture, when adhesion reached a plateau during time-course experiments. The composite tracings of Figure 1
(lower panel) depict the progressive elevation of Ca2+-dependent fluorescence in monolayers co-cultured with U937 cells in comparison with time-control monolayers (Figure 1
, upper panel). The time course of [Ca2+]i increases in HMC exposed to U937 cells is shown in Figure 2
, with a significant elevation as early as 60 min, reaching a plateau at 180 min, with a further slight rise at 360 min. Of interest, progressive insensitivity to the Ca2+-mobilizing effects of angiotensin II (ANG II) was apparent even at early time points in co-cultured HMC, while control monolayers retained complete responsiveness to the octapeptide, as shown by the sharp [Ca2+]i transient in panel A at 180 min. Figure 3
summarizes the timing of downregulation of the [Ca2+]i responses to 1 µM ANG II, with complete suppression after 360 min of the [Ca2+]i elevation resulting from combined release of Ca2+ from inositol (1,4,5)-trisphosphate (InsP3)-sensitive stores and Ca2+ influx through membrane channels [14,18,19]. Similar experiments were carried out on PBM freshly isolated from healthy donors, resulting in a nearly identical time-course of resting [Ca2+]i elevation and progressive desensitization to the effects of ANG II (Table 1
). Thus, the effects herein observed are not unique to the U937 cell line, possibly as a result of neoplastic cell dedifferentiation, but are rather shared by normal monocytes and thus truly representative of this leukocyte subset. Inhibition of [Ca2+]i signalling in response to vasoconstrictors was not restricted to ANG II, nor to a depletion of Ca2+ stores as in the case of inhibition of uptake or slow outflow. Table 2
compares to this end the response to another receptor-mediated stimulus of [Ca2+]i, the thromboxane A2 (TxA2) analogue, U-46619, with the effects of two structurally unrelated Ca2+ ionophores, ionomycin and 4-Br-A23187. As can be seen, while U-46619 failed to elicit a [Ca2+]i transient at any tested concentration, similar to ANG II, both ionophores promptly released normally repleted Ca2+ stores. Prior application of either ionophore prevented subsequent Ca2+ mobilization by ANG II or U-46619 (e.g. [Ca2+]i increases after 5 µM ionomycin +22±6 and +16±5% respectively above baseline, P=NS.

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Fig. 1. [Ca2+]i monitoring in fura 2-loaded HMC monolayers during 6-h co-culture with 1x106 U937 cells/ml (lower panel). Note progressive increase of [Ca2+]i compared with control monolayers (upper panel), and loss of responsiveness to 1 µM angiotensin II (ANG II) at 180 min. Composite fluorometric tracings representative of n=12 experiments.
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Fig. 2. Average [Ca2+]i levels at timed intervals in fura 2-loaded HMC monolayers during 6-h co-culture with 1x106 U937 cells/ml. Note progressive increase of [Ca2+]i. *P<0.05, **P<0.01 vs basal [Ca2+]i, one-way ANOVA on n=12 experiments.
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Fig. 3. Time-course of desensitization of the [Ca2+]i response to 1 µM angiotensin II (ANG II) in fura 2-loaded HMC monolayers during 6-h co-culture with 1x106 U937 cells/ml. Average [Ca2+]i levels at timed intervals. *P<0.05, **P<0.01 vs [Ca2+]i peak at 0 time (basal), one-way ANOVA on n=12 experiments.
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Table 1. Effects of co-culture with peripheral blood monocytes (PBM) on basal and angiotensin II (ANG II)-stimulated [Ca2+]i in fura 2-loaded HMC monolayers
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Table 2. Effects of co-culture with U937 cells on the [Ca2+]i response to vasoconstrictors or Ca2+ ionophores in fura 2-loaded monolayers of HMC
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The effects of co-culture with U937 cells were not attributable to a diffusible mediator released in the bathing media, as both conditioned media from U937 cells and from co-cultured HMC+U937 cells failed to elicit any change of [Ca2+]i in HMC (Table 3
). To account for the possible short-life of the putative released factor(s), immediate freezing of the culture supernatants was ensured, with subsequent thawing and immediate addition to the HMC monolayers. Another approach taken to rule out a soluble mediator was the physical separation of U937 from HMC monolayers by plating the former onto plastic 0.45 µm porous filter inserts bathed in the same experimental medium. These filters allow for free diffusion of cell products, preventing at the same time any contact between the two cell populations studied. Table 4
shows that this protocol too failed to affect HMC [Ca2+]i and responses to ANG II. Thus, physical contact between the two cell types, i.e. monocyte adhesion, was required to induce the above described response in HMC, which was not mimicked by cytokines, growth factors or prostanoids possibly released during co-culture.
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Table 3. Effect of conditioned media from U937 cultures and HMC+U937 co-cultures on [Ca2+]i of cultured HMC monolayers
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Table 4. Effects of co-culture with U937 cells applied onto MillicellTM-HA 0.45 um culture plate inserts on baseline and angiotensin II (ANG II)-stimulated [Ca2+]i in fura 2-loaded monolayers of HMC
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The other arm of the interplay between HMC and monocytes was examined as well. Figure 4
depicts an identical protocol in suspensions of U937 cells selectively loaded with fura 2. These cells retained baseline Ca2+ values throughout the entire length of 6-h co-cultures with HMC, along with normal sensitivity to the specific phospholipase C agonist, adenosine (3',5')-trisphosphate (ATP), which rapidly stimulated the release of intracellularly stored Ca2+ (lower panel).

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Fig. 4. [Ca2+]i monitoring in suspensions of fura 2-loaded U937 cells (1x106 cells/ml) during 6-h co-culture with confluent HMC monolayers (lower panel). Note stable [Ca2+]i throughout the observation in both co-cultured and control U937 cell suspensions (upper panel), and normal responsiveness to 1 mM ATP at 180 min. Composite fluorometric tracings representative of n=9 experiments.
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In order to identify the cellular mechanism by which the interaction with U937 cells increases [Ca2+]i in cultured HMC, we repeated a set of experiments in nominally Ca2+-free solutions. A preliminary evaluation of monocyte binding demonstrated substantial adhesion even under Ca2+-free conditions, with an actual modest increase of the ratio U937 cells/HMC to 1.1±0.1 (P=NS vs 1.25 mM Ca2+). As shown in Figure 5
, fluorometric monitoring resulted in steady [Ca2+]i after a brief persistent decrease upon switching to Ca2+-free media, likely due to Ca2+ efflux down an outward gradient. The new baseline levels were then maintained for 360 min in the continued presence of U937 cells. At any time point during the experiment, addition of extracellular Ca2+ resulted in a rapid rise of [Ca2+]i through surface channels which are activated by Ca2+ depletion. As previously reported, these channels belong to the functional group of store-operated Ca2+ channels (SOC), responsible for the so-called capacitative Ca2+ influx, since it enables refilling and subsequent discharge of InsP3-sensitive cytoplasmic stores [1820]. In Ca2+-free solutions, HMC did not lose their sensitivity to ANG II even at 180 (lower panel) or 360 min. Therefore, Ca2+ influx appears to be the mechanism that underlies the persistent elevation of [Ca2+]i in HMC co-cultured with U937 cells. To further confirm that internalization of Ca2+ is necessary to support the signalling changes observed in HMC, we substituted Mn2+ for Ca2+ in the bathing medium, monitoring the fluorescence decay resulting from irreversible quenching of fura 2 (Figure 6
). In 6-h co-cultures, the slope of Mn2+-quenching was constantly steeper than in control HMC cultures, with obvious desensitization of the response to ANG II. As any vasoconstrictor mobilizing Ca2+, ANG II abruptly increased the rate of fluorescence extinction in control cultures (upper panel), due to opening of non-selective divalent cation channels, with resulting enhanced entry of Mn2+. This effect was lost in the co-cultures (lower panel), suggesting maximal increase of divalent cation conductance as the key mechanism for the observed effect of monocytes on target HMC.

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Fig. 5. [Ca2+]i monitoring in fura 2-loaded HMC monolayers during 6-h co-culture with 1x106 U937 cells/ml in Ca2+-free media (lower panel). Note immediate [Ca2+]i decrease upon addition of Ca2+-free media, and stable [Ca2+]i in both co-cultures and control monolayers (upper panel) throughout the observation. Also shown stable response to addition of 1 mM Ca2+ at 60 min and 1 µM angiotensin II (ANG II) at 180 min. Composite fluorometric tracings representative of n=6 experiments.
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Fig. 6. Mn2+-quenching of fura 2 fluorescence in HMC monolayers during 6-h co-culture with 1x106 U937 cells/ml (lower panel). Note progressive Mn2+ influx into co-cultured HMC, revealed by accelerated decay of fura 2 fluorescence, compared with the more stable emission of control monolayers (upper panel), and partial loss of responsiveness to 1 µM angiotensin II (ANG II) at 60 min. Fluorometric tracings representative of n=6 experiments.
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Discussion
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The interaction between leukocytes and resident cells is a key event in renal inflammation [15,22]. In vitro techniques have shown a remarkable potential for the study of this interplay, which is also a sensible target for the therapy of acute and chronic renal disease [7,2126]. Our approach relies on well established monocytoid cells of the U937 line, which share most characteristics of PBM, without the intra- and interindividual variability intrinsic to preparations from multiple donors on a day-by-day basis [811]. We have previously shown that U937 cells and PBM share a common behaviour in adhering to monolayer cultures of HMC without need for priming by cytokines or prior differentiation into macrophages. The process is driven by adhesion molecules expressed by HMC, recognized by counterreceptors of the LFA-1 and VLA-4 types on both U937 and PBM [4,8,9,23]. Cytokines and certain vasoactive agents upregulate the process of adhesion without relevant differences in the profile of receptors expressed on either cell population. For these reasons, U937 cells have been extensively used for the study of differentiation of the monocytic/macrophage lineage, along with the biochemistry of lymphoid cells [27]. In our co-culture experimental setup, they provide a reliable means of assessing the interplay of circulating, non-activated monocytes with resident cells of the human glomerulus, with ease of manipulating the growth conditions so as to mimic different disease microenvironments. Indeed, in the present study, U937 cells and PBM once again confirm their functional identity in binding to HMC, resulting in similar changes of [Ca2+]i after some time in co-culture. It is noteworthy that time-dependency of the response and downregulation of the effects of ANG II on HMC are remarkably similar for both monocytic cell lines.
The present data identify a mechanism by which HMC sense binding of U937 cells, with resulting offsetting of other signals such as those elicited by vasoconstrictors, namely ANG II. This is not unprecedented in other cell types, since cell-mediated immune responses are often accompanied by a rise of [Ca2+]i in the effector cell, followed by a slow elevation in target cells resulting from a passive leak across the damaged cell membrane [12,13,20]. Yet, to our knowledge this is the first evidence of such interaction occurring at the level of glomerular mesangial cells, upon adhesion of undifferentiated monocytoid cells not expressing the phenotype of activated cytotoxic NK lymphocytes. The relevance of this finding is twofold: first, non-activated monocytes homing into mesangial spaces under normal conditions may elicit transmembrane signals in HMC, likely translating into adaptive responses aimed at regulating monocyte influx and preventing in situ activation; second, larger immigration of monocytes at sites of chronic glomerularor tubulointerstitialinflammation may elicit massive and persistent increments of [Ca2+]i in HMC. This latter setting is more closely duplicated by our technique, which places large numbers of monocytes, i.e. 1x106 cells/ml, in a stirred suspension in constant contact with the HMC monolayer, an event that is followed within minutes by the persistent adhesion of a limited number of activated monocytes to the surface of the glomerular cells (i.e. 12 per each individual HMC). Taking into account the fact that the [Ca2+]i signal revealed by our fluorometric technique is an average emission from several cells in the optical path, the possibility exists that certain cells have brisker [Ca2+]i elevations, while others do not modify or just slightly increase their baseline levels throughout the incubation, leading to underestimation of the overall response. An interesting issue that awaits evaluation is whether uneven responses can be detected at the single cell level between the HMC binding U937 cells and those only exposed to diffusible mediators released in the bathing medium. To account for the possible effects of cytokines or growth factors released by U937 cells during long-term incubations, we carried out control experiments employing pooled conditioned media from monocytes or monocyte/HMC co-cultures, as well as U937 cells co-cultured on culture plate inserts preventing contact between the two cell populations. These experiments addressed the possibility that stable or even labile compound(s), such as a prostanoid active on [Ca2+]i (e.g. prostaglandin F2
, TxA2, leukotrienes C4/D4) released by U937 cells might act on HMC to elicit the slow Ca2+ changes observed [14]. The data unequivocally indicate that conditioned media have no effects on [Ca2+]i of HMC, and therefore the observed response to the co-culture with U937 cells must be considered to be mediated by the physical interaction between the two cell lines.
The fura 2 fluorometric technique allowed the selective loading of either cell population to monitor Ca2+ fluxes during co-culture, clearly showing that no [Ca2+]i changes occur in U937 cells in response to the interaction with HMC. Similarly, the physical perturbation due to continuous stirring of the suspension and the shear stress due to random contact with the HMC monolayer resting on a plastic coverslip did not affect baseline [Ca2+]i levels of the U937 cells. This should not be taken as absolute evidence of the lack of involvement of [Ca2+]i signalling in monocyte adhesion to HMC. It should be considered that the absolute number of adherent cells is negligible when compared to the total mass of monocytes employed (few hundred/coverslip vs 1x106/ml), so that a possible increase of [Ca2+]i in individual cells adherent to HMC would be diluted in a much larger population of freely-floating U937 cells with steady [Ca2+]i levels.
Concerning the mechanism by which co-culture results in an elevation of [Ca2+]i, our studies provide evidence for passive entry through plasma membrane channels, since removal of extracellular Ca2+ both abolishes the slow rise of [Ca2+]i and preserves sensitivity of the monolayers to the action of a Ca2+-mobilizing agent such as ANG II. The theoretical possibility that Ca2+-free media could reduce binding of monocytoid cells, thus decreasing any effect on HMC [Ca2+]i, was ruled out by direct cell counts showing that U937 cell adhesion is independent of ambient [Ca2+]. Should slow release of stored Ca2+ be responsible, the phenomenon would be self-limiting, because of exhaustion of stores and Ca2+ extrusion by otherwise functional plasma membrane Ca2+ pumps [15,1820]. Additionally, Ca2+ withdrawal from the bathing medium would allow complete depletion of the Ca2+ stores, making it impossible for ANG II to trigger the responses observed at 180 min in Figure 1
. Among the possible channels involved, our attention focused on the so-called store-operated Ca2+ (SOC) influx pathway, also referred to as capacitative Ca2+ influx [15,1820]. In our experimental conditions this Ca2+ conductance activated by previous discharge of intracellular Ca2+ stores or by the blocker of the endoplasmic reticulum Ca2+-ATPase, thapsigargin, is by far the most prominent divalent cation entry route for HMC [15,1820]. Both ionophores and receptor-transduced agents that release InsP3 from membrane phosphoinositides share the ability of increasing the conductance of such channels, which are typically insensitive to dihydropyridines or blockers of conventional voltage-operated Ca2+ channels (VOC). The latter are hardly detectable in our monolayer cultures, as changes of [Ca2+]i have not been consistently reported in response to depolarization or application of Bay K 8644, an agonist of VOC in smooth muscle cells and other contractile cells [15,19,28,29]. Of course, a passive Ca2+ leak pathway cannot be discounted, in view of previously published evidence of cytotoxic damage to HMC by monocytes/macrophages, including co-cultured U937 cells in earlier studies [5,8,9]. Pore-forming products of monocytes may also be considered, similar to other models of cell-mediated cytotoxicity [7,12,13]. However, the Ca2+-free experiments tend to rule out a form of dysregulated Ca2+ transport, as rapid depletion of cytosolic Ca2+ would ensue in the absence of extracellular Ca2+ as a result of outflow down a reverse gradient across a damaged plasma membrane. Therefore, the inverse relationship, that is, a slow increase of [Ca2+]i leading to cell injury, apoptosis, or both, is more likely, particularly considering the similar time-frame of LDH release and evidence of cell damage [9].
A rather prompt downregulation of the response to ANG II is another relevant feature of HMC exposed to U937 cells. This is not unique to the co-culture setting, however, since almost any agent increasing [Ca2+]i downregulates the response to the same or unrelated agonists [14,19,28,29]. Homologous desensitization is under most circumstances related to receptor saturation following the first application of a stimulus. Only agents with rapid dissociation kinetics can activate again the cells within minutes from washing away the bathing medium. Heterologous desensitization, on the other hand, describes the inability of an agent to elicit a [Ca2+]i response following application of one or more unrelated stimuli [14,15,28]. This is typically the case of fetal serum used in cell culture, containing a variety of vasoconstrictors resulting from the platelet release reaction, which must be withdrawn at least 12 h prior to measurement of [Ca2+]i in order to detect significant responses to any agent tested [14,28]. We show herein that Ca2+ ionophores duplicate this downregulation, obviously by a mechanism not directly involving receptors. Depletion of InsP3-sensitive Ca2+ stores, activation of protein kinase C by Ca2+ itself, feedback inhibition by Ca2+ of gated Ca2+ stores, coupling G-proteins and/or receptors are all potential mechanisms that likely cooperate in dampening sensitivity of the cells following sequential exposure to vasoconstrictors [14,15,18,19]. The significance of the process is probably to limit the extent of cell activation by [Ca2+]i agonists through a refractory interval. It is of interest that monocytes express such inhibitory activity on ANG II signalling, as this may be relevant to more distal events thought to participate in the regulation of renal haemodynamics, including glomerular filtration, proteinuria, glomerulosclerosis and interstitial fibrosis driven by this octapeptide.
Taken together, these data provide the biochemical basis of an intercellular relationship between blood-borne cells and resident glomerular cells. This cross-talk translates into functional responses, including HMC cytotoxicity and reactive proliferation, cytokine release and further monocyte chemoattraction, enhanced collagen IV and fibronectin biosynthesis recently observed in high glucose media [30]. The relative contribution of [Ca2+]i signalling to the interaction between monocytes and HMC still awaits elucidation by comparison with other potentially involved signals, such as the interaction between leukocyte counterreceptors and membrane-anchored mesangial immunoglobulins of the adhesion molecule superfamily. Work at the single cell level in vitro and in vivo should eventually shed light on the direct events occurring at the interface between infiltrating monocytes and kidney cells in health and disease.
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Acknowledgments
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These studies were supported by grants from the Ministero della Università e Ricerca Scientifica (quote 40 and 60%) to F.P. and G.A.C.
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Notes
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Correspondence and offprint requests to: Paolo Menè, Division of Nephrology, Department of Clinical Sciences, Policlinico Umberto I, Viale del Policlinico 155, I-00161 Rome, Italy. Email: Paolo.Mene{at}uniroma1.it 
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Received for publication: 13. 1.01
Revision received 11. 9.01.