Assessment of Rapid Morphological Changes Associatedwith Elevated cAMP Levels in Human Orbital Fibroblasts

Lavanya Reddy,* Hwai-Shi Wang,†,1 Charles R. Keese,* Ivar Giaever,* and Terry J. Smith†,2

*School of Science, Rensselaer Polytechnic Institute and Applied BioPhysics, Inc., Troy, New York 12180; and †Division of Molecularand Cellular Medicine, Department of Medicine and Department of Biochemistry and Molecular Biology, Albany Medical College

and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208

Orbital fibroblasts exhibit a phenotype distinct fromthat of other types of fibroblasts. Addition of prosta-glandin E2 (PGE2) to culture medium elicits a dra-matic change in orbital fibroblast morphology. Thatresponse is mediated through the generation of cAMP.Orbital fibroblasts can generate high levels of PGE2

through induction by proinflammatory cytokines ofprostaglandin endoperoxide H synthase-2 (PGHS-2). Herewe compare the influence on fibroblast morphology ofexogenous PGE2, forskolin, and 8-br-cAMP to that me-diated through PGHS-2 induction by a lymphocyte-derived cytokine. Within a few hours, orbital fibroblaststreated with any of these test compounds appear un-der phase-contrast microscopy to exhibit a stellatemorphology. When these changes were assessed quan-titatively by electric cell–substrate impedance sensing(ECIS), it became evident that 8-br-cAMP, forskolin,and PGE2 initiated shape changes within 30 min ofaddition to the culture medium, while effects of thecytokine were first evident after approximately 3.5 h.Dermal fibroblasts failed to respond to any of thesecompounds with regard to changes in cellular mor-phology. Analysis of micromotion, manifested as smallimpedance fluctuations, revealed that orbital fibro-blasts treated with 8-br-cAMP exhibit less motion thandid untreated cells. These results suggest that orbitalfibroblast shape can be altered by several compoundsknown to alter intracellular cAMP levels. They dem-onstrate the utility of ECIS in the assessment of veryrapid and dynamic cellular events associated withchanges in cell morphology. © 1998 Academic Press

Key Words: Graves’ disease; thyroid associated oph-thalmopathy; electric cell–substrate impedance sens-ing; cytokine action.

INTRODUCTION

Fibroblasts represent a diverse population of cells.Despite the perception that fibroblasts function pri-marily to support more specialized cells, an emergingbody of evidence suggests that they are an importantsite of small regulatory molecule biosynthesis. More-over, fibroblasts appear capable of dynamic responsesto extracellular signals in the context of tissue stressand reorganization such as occurs in inflammation. Wehypothesize that the phenotypic attributes manifestedby particular populations of fibroblasts reflect the pe-culiar roles those cells play in the tissue of origin.Moreover, differences among fibroblasts may accountfor the susceptibility to disease associated with certainanatomic regions. Fibroblasts from the synovium [1],lung [2], and orbit [3] exhibit particular susceptibilityto proinflammatory signals. Thus, the propensity ofthese areas of the human body to manifest systemicdisease may be a consequence, at least in part, of thephenotype engendered by fibroblasts inhabiting thosetissues.

Orbital fibroblasts are derived from the neural ecto-derm and exhibit distinct phenotypic characteristics invitro [4]. While resembling most other fibroblasts atthe cytoarchitectural level [5], they have a morphologydistinct from that of dermal fibroblasts [6] and responddifferentially to a number of proinflammatory cyto-kines [3, 7, 8]. Their patterns of receptor, ganglioside,and glycosaminoglycan expression and responses to avariety of hormones, cytokines, and other small regu-latory molecules differ from those of other fibroblasts[3, 7–15]. These fibroblasts have a putative role in thepathogenesis of thyroid-associated ophthalmopathy(TAO), an autoimmune disease associated with a dra-matic remodeling of the orbital connective tissues [16].It is currently believed that fibroblast activation is acritical component of the pathogenesis of TAO.

We have reported recently that prostaglandin E2(PGE2) elicits a rapid and transient change in themorphology of cultured orbital fibroblasts from pa-tients with severe TAO [9, 13]. In contrast, dermal

1 Present address: Department of Anatomy, School of Life Science,National Yang-Ming University, Taipei, Taiwan.

2 To whom correspondence and reprint requests should be ad-dressed at Division of Molecular and Cellular Medicine (A-175),Albany Medical College, 47 New Scotland Avenue, Albany, NY12208. Fax: (518) 262-5304.

3600014-4827/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

EXPERIMENTAL CELL RESEARCH 245, 360–367 (1998)ARTICLE NO. EX984273

fibroblasts do not undergo a change in morphology inresponse to the prostanoid [13]. The transition from atypical fibroblast-like shape to a stellate shape is veryrapid. PGE2 and its analogues affecting cell shapestimulate endogenous cAMP production, while prosta-noid analogues that did not influence cell shape failedto activate cyclic nucleotide generation [9]. We havealso observed that prostaglandin endoperoxide H syn-thase-2 (PGHS-2), the inflammatory cyclooxygenase, isparticularly inducible by the proinflammatory cytokineleukoregulin in orbital fibroblasts [3]. This induction ofPGHS-2 is mediated at both transcriptional and post-transcriptional levels and results in the generation ofsubstantial levels of PGE2 and cAMP [3]. The synthesisof PGE2 and cAMP can be blocked by glucocorticoidswhich attenuate PGHS-2 expression and by the PGHS-2-selective inhibitor SC 58125 [3].

In this paper, we examine the very rapid changes inmorphology that occur when orbital fibroblasts aretreated with 8-bromoadenosine-39,59-cyclic monophos-phate (8-br-cAMP) or compounds that increase endog-enous cAMP generation through the activation of ade-nylate cyclase, such as PGE2 and forskolin. These arecompared with those elicited through an induction ofPGHS-2 and the resultant endogenous prostaglandinproduction. We assessed cell morphology with phase-contrast microscopy and with electric cell–substrateimpedance sensing (ECIS) [17–20]. The later techniqueoffers insight into subtle, rapid changes not apparentwith light microscopy. ECIS thus has substantial util-ity in determining the kinetics of rapid cellular re-sponses culminating in altered cell morphology. Theresults suggest that several molecular signals leadingto cAMP generation are capable of eliciting rapid shapechange in orbital fibroblasts. By using ECIS we areable to distinguish differences in the rates of changeelicited by leukoregulin, requiring activation of geneexpression, from those not involving de novo proteinsynthesis.

MATERIALS AND METHODS

Dubecco’s modified Eagle’s medium (DMEM) and fetal bovine se-rum (FBS) were purchased from GIBCO. PGE2 and 8-br-cAMP wereobtained from Sigma (St. Louis, MO); dibutyrladenosine-39,59-cyclicmonophosphate (dibutyrl cAMP) was obtained from ICN Biomedicals(Aurora, OH) and forskolin was from Calbiochem (La Jolla, CA).Electrodes used in this study were fabricated by Applied Biophysics,Inc., as described previously [13, 19]. Leukoregulin was kindly sup-plied by Dr. Charles Evans (NCI).

Cell culture. Orbital fibroblasts were obtained from patients withor without Graves’ disease at the time of orbital surgery. Dermalfibroblasts were obtained from biopsies of normal-appearing abdom-inal skin. Institutional review board approval at the Albany MedicalCollege was obtained to conduct these studies. The tissue biopsieswere grown on plastic tissue culture dishes bathed in Eagle’s me-dium supplemented with 10% FBS and antibiotics as described pre-viously [21]. The cultures were maintained in a humidified 5% CO2

incubator at 37°C and passaged serially using trypsin/EDTA. LiquidN2 was used in the long-term storage of some cultures. Medium waschanged every 3–5 days and cells were not used beyond passage 14from culture initiation. In these experiments, cultures that served ascontrols received an equivalent aliquot of fresh medium at the sameinstant as those receiving test compounds were manipulated. A totalof at least six orbital (three from patients with TAO and three fromnormal connective tissue) and six dermal strains were examined inthese studies. Each came from a different donor. The characteristicfindings peculiar to TAO, normal orbital and dermal fibroblasts werefound consistently in all strains tested.

Phase-contrast microscopy. Fibroblasts were inoculated in wellshaving a base area of 0.5 cm2 at a density of 2 3 105 cells/cm2. Thecells were allowed to attach and spread for 2–3 days prior to anyexperimental manipulations. 8-br-cAMP (4 mM) or an equivalentvolume of DMEM was added to the wells and observed under themicroscope 6 h later. Cells were fixed in 3% formaldehyde in phos-phate-buffered saline (PBS, pH 7.3) and then washed three timeswith PBS. Microscopy was performed using a Nikon 35-mm cameraand Kodak Royal Gold 400 film at a final magnification of 3400. Allcells shown in the photographs were representative of the particularculture in which they were cultivated.

Impedance measurement by ECIS. The impedance of fibroblastsmaintained as a monolayer in culture was measured using a tech-nique described previously [9, 19, 22]. In this system, cells weregrown on small gold electrodes and the culture medium was used asan electrolyte. An AC signal of 1 mA, supplied by an approximatelyconstant current source, was applied between a small active elec-trode (5 3 1024 cm2) and a larger counter electrode. The voltage wasmonitored by a lock-in amplifier and a personal computer stored andprocessed voltage and phase data as well as controlling the setting ofthe amplifier.

The electrode arrays in these studies consisted of five wells (0.5cm2 base area), each of which was mounted on a common base. Thesewere connected to a relay bank that was interfaced to a PC such thatmeasurements could be changed from one well to another during thecourse of an experiment. As cells attach and spread on the goldelectrodes, their plasma membranes act as insulators and block thecurrent path. This forces the current to flow around and between theindividual cells [17]. The impedance, measured using a 4000-Hzsignal, is usually reported as a resistance and capacitance by theECIS software, treating the system as a series RC circuit.

RESULTS

When maintained in cell culture, human fibroblastsassume a characteristic shape regardless of their ana-tomic site of origin (Fig. 1A). It is only under closeinspection that modest differences can be detected atthe level of light microscopy [6]. Moreover, the cytoar-chitecture of human fibroblasts from different sub-populations is nearly identical when examined withtransmission electron microscopy [5]. Orbital fibro-blasts from patients with TAO, treated with either8-br-cAMP (4 mM) or dibutyrl cAMP (4 mM), undergoa dramatic shape change. As is demonstrated in Fig.1B, fibroblasts treated with either compound for 6 hdevelop a stellate shape with multiple processes ex-tending from the central part of the cells. The centralnucleus containing part of these cells appears slightlyraised above the plane of the substratum comparedwith cells not undergoing shape change. In contrast,dermal fibroblasts treated with these compounds fail to

361MORPHOLOGY CHANGES IN ORBITAL FIBROBLASTS

exhibit shape changes. The appearance of orbital fibro-blasts treated with 8-br-cAMP is indistinguishablefrom that of cells treated with exogenous PGE2. Thepopulation of fibroblasts treated with 8-br-cAMP iscomposed of individual cells that vary extensively interms of their morphology. Some cells develop long,distinct processes that range in number from 5 to 25.

Others appear to remain relatively unchanged and ex-hibit few processes. In some of the cells undergoing anextensive change in morphology, small, thin secondaryprotrusions extend from many primary processes.

Orbital fibroblasts from normal connective tissueand abdominal dermal fibroblasts were also treatedwith 8-br-cAMP under identical conditions. Normal or-

FIG. 1. Phase-contrast micrographs of orbital fibroblasts from patients with TAO (A,B), normal orbital connective tissue (C,D), anddermal fibroblasts from the abdominal wall (E,F). Cultures were treated with nothing (A,C,E) or with 8-br-cAMP (4 mM) (B,D,F) for 6 h.

362 REDDY ET AL.

bital fibroblasts (Fig. 1D) respond in a qualitativelysimilar manner to those from donors with TAO. How-ever, the proportion of normal orbital fibroblasts un-dergoing shape change is significantly less than thatobserved in TAO orbital fibroblasts. In a typical exper-iment, approximately 80% of orbital fibroblasts fromdonors with TAO develop many distinct processes com-pared with 30% in the normal fibroblast cultures. Un-treated normal orbital fibroblasts (Fig. 1C) have a mor-phology that is indistinguishable from that observed inthe control TAO cultures. Abdominal dermal fibro-blasts, in contrast, fail to respond to 8-br-cAMP. Themorphology of the treated dermal fibroblasts (Fig. 1F)is indistinguishable from that of untreated dermal fi-broblasts (Fig. 1E).

We next examined the effects of 8-br cAMP anddibutyrl cAMP on cell shape with ECIS. Responses tothese compounds were identical and thus they wereused interchangeably. Dibutyrl cAMP (4 mM) causes atime-dependent drop in the monolayer resistance oforbital fibroblasts from patients with TAO (Fig. 2, top).The decrease occurred within 30 min of addition of thecompound to the culture medium and was sustainedfor the duration of the experiment (4 days). The ex-pected increase in monolayer capacitance followingtreatment accompanied the drop in resistance (datanot shown). Dermal fibroblasts from the abdominalwall failed to respond to exogenous cAMP, as is evi-denced by the failure of 8-br-cAMP to alter monolayerresistance (Fig 2, bottom). PGE2 (1 mM) causes a de-crease in the monolayer resistance, but, unlike theeffects seen with cAMP analogues, the effects of PGE2are transient, lasting approximately 10 h before re-turning to control values. Forskolin (20 mM) alsocauses a drop in the monolayer resistance of orbitalfibroblasts from donors with TAO following its additionto the culture medium (Fig. 3). The magnitude of thiseffect was virtually the same as was seen with exoge-nous cAMP (Fig. 2). Forskolin was tested because itdirectly stimulates the generation of cAMP by actingon adenylate cyclase.

Induction of PGHS-2 by proinflammatory cytokines,including leukoregulin and IL-1b, can lead to substan-tial increases in PGE2 production in TAO orbital fibro-blasts [3]. When orbital fibroblasts were exposed toleukoregulin (1 U/ml), a change in morphology occursthat is similar to that with 8-br-cAMP and PGE2 treat-ment. Within 3 h, a large proportion of the cells havebegun to develop multiple cytosolic processes. Whenassessed by light microscopy, the kinetics of thechanges in morphology elicited by leukoregulin wereindistinguishable from those of the other compounds.We therefore utilized ECIS to further characterizethese shape changes. We compared the effects of leu-koregulin with those of PGE2 on monolayer impedance.As Fig. 4 suggests, there is a considerable difference in

the time course in which resistance decreases followingtreatment with PGE2 and the cytokine. In the case ofthe prostanoid, resistance decreases within 30 min ofaddition to the culture medium, while the effect ofleukoregulin is delayed. Its effects evolve between 2.5and 4.5 h. The response to leukoregulin is dose-depen-dent in the range of 0.1–1 U/ml (Fig. 5).

The rapid resistance fluctuations observed in thedata curve sets shown in Figs. 2–4 are a result of themicromotions of cells in confluent layers [17]. We notedthat the amplitude of micromotion in cells treated with8-br-cAMP, leukoregulin, and the other compounds el-evating cAMP levels was reduced. We then set out todetermine whether this observation was consistent

FIG. 2. (Top) Time course of the effect of dibutyrl cAMP onnormalized resistance in orbital fibroblasts from a patient with TAO.(Bottom) Time course of the effect of 8-br-cAMP on normalized re-sistance in dermal fibroblasts. Confluent monolayers of fibroblastswere shifted to fresh medium without or with the cAMP analogue (4mM) at the time designated “0.” Resistance was assessed using ECISas described under Materials and Methods. Normalized resistancewas determined by dividing the resistance at the various times bythe resistance measured at time “0.” The tracings shown are fromsingle, representative experiments.

363MORPHOLOGY CHANGES IN ORBITAL FIBROBLASTS

and characteristic of orbital fibroblasts from patientswith TAO or generalizable to other fibroblasts as well.Using ECIS, this cellular micromotion was monitoredin monolayers of TAO orbital fibroblasts and normalorbital and dermal cultures under basal conditions andfollowing treatment with 8-br-cAMP (4 mM). The av-erage variance calculated for 64 time segments of 64-sintervals for untreated TAO orbital fibroblasts shownin Fig. 6 is 79.8 ohm2, while the average variance in8-br-cAMP-treated cultures is 9.4 ohm2, representing

12% of the control value. These differences betweencontrol and treated cultures are not as striking innormal orbital fibroblasts. The average variance inresistance is 105.4 ohm2 in untreated cells and 38.8ohm2 in cultures receiving 8-br-cAMP, which is 36% ofthe control variance. Thus, untreated orbital fibro-blasts, regardless of whether from donors without orwith TAO, exhibit an average variance that is substan-tially greater than that observed in 8-br-cAMP-treatedcultures. In contrast, the average variance in un-treated dermal fibroblasts is 13 ohm2, while the aver-age variance in treated cultures is 16.2 ohm2, which is125% of the control value. Figure 7 summarizes theaverage 64-point variance in resistance found in TAOand normal orbital and dermal fibroblasts under un-treated and 8-br-cAMP-treated culture conditions.

DISCUSSION

Fibroblasts derived from the human orbit appear tobe distinct from fibroblasts of extraorbital origin. Or-bital fibroblasts are particularly susceptible to the ac-tions of proinflammatory cytokines such as interfer-on-g, TGF-b, IL-1b, and leukoregulin when comparedto other types of fibroblasts [3, 7, 8, 10, 11, 14, 23]. Thephenotypic differences setting orbital fibroblasts apartfrom other fibroblasts may play a role in the site-selective vulnerability of the human orbit to disease.Moreover, these differences likely reflect the uniquephysiologic roles played by orbital fibroblasts.

PGE2 and other compounds that increase cAMP lev-els elicit in orbital fibroblasts a rapid and dramaticshape change [9, 13]. In contrast, dermal fibroblasts

FIG. 3. Time course of the effect of forskolin on normalizedresistance of orbital fibroblasts. Confluent monolayers of TAO fibro-blasts were shifted to fresh medium without or with forskolin (20mM) at time “0.” Measurements were taken as described in thelegend to Fig. 1.

FIG. 4. Comparison of the time course of effects on normalizedresistance elicited by PGE2 and the proinflammatory cytokine, leu-koregulin. At time “0,” monolayers were shifted to fresh mediumcontaining nothing, PGE2 (1 mM), or leukoregulin (1 U/ml). Thetracing was from a single, representative experiment.

FIG. 5. Dose–response of the leukoregulin effects on normalizedresistance in TAO orbital fibroblasts. Confluent monolayers wereshifted to fresh medium containing nothing (control) or the gradedconcentrations of leukoregulin indicated at time “0.” Normalizedresistance was then assessed as described in the legend to Fig. 1.

364 REDDY ET AL.

are considerably less responsive to the shape alterationresulting from increases in cAMP. This cellular re-sponse appears most robust in TAO orbital fibroblasts;however, it is also present in normal orbital fibroblasts.The extent to which orbital fibroblasts respond to ele-vated cAMP levels may reflect differences in the cAMPgeneration cascade. Differences in PGE2 surface recep-tor density could underlie the exaggerated response tothe prostanoid. Our finding, however, that TAO fibro-blasts are also more responsive to exogenous cAMPsuggests rather that differences may reside in eventsdownstream of cyclic nucleotide generation. Thesecould involve cell-specific differences in the relevantsignal transduction cascades utilized by the shape-altering compounds. Alternatively, the levels of actinor actin-stabilizing proteins could differ in varioustypes of fibroblasts or qualitative differences in theseor other key structural proteins might exist. Clearly,future studies will need to be directed at defining themolecular basis for the differential sensitivities toagents that modify cellular shape exhibited by TAOorbital fibroblasts.

The change in cell morphology due to elevated cAMPlevels results in a stellate shape which resembles ef-fects reported previously in various cell types. Synovialcells from rheumatoid synovitis have been described asbeing stellate in shape [24]. Langerhans cells and pe-ripheral blood dendritic cells of the immune system arealso stellate in shape [25]. However, there is currentlyno evidence to suggest that orbital fibroblasts whichbecome stellate in shape acquire any special immuno-logic functions. Jumblatt and co-workers have demon-strated the ability of PGE2 to regulate corneal endo-thelial shape change [26, 27]. The shape change evokedby the prostanoid in this case involves binding to anEP2 class receptor and production of cAMP [27]. Cy-tochalasins and lovastatin induce shape changes sim-ilar to those caused by elevated cAMP levels in cul-tured aortic smooth muscle cells [28–30]. Cells offibroblastic lineage adopt stellate shapes duringspreading on fibronectin in the presence of compoundswhich increase cAMP levels [31]. The extended pro-cesses stained strongly for F actin and time-lapse videorecording demonstrated that the stellate shapes weregenerated by outgrowths of neurite-like processes. Thedepletion of microfilaments, stress fibers in particular,accompanies stellation that results directly from ele-vated cAMP levels [29, 32, 33]. These findings supportthe concept that stellate shapes are caused by thedepletion of actin microfilaments.

Changes in monolayer resistance that result fromshape alteration could be detected by ECIS within 30min after the test compounds were added to the culturemedia. This was well in advance of the earliest changes

FIG. 6. Effect of 8-br-cAMP on cellular micromotion in fibro-blasts from TAO and normal orbital connective tissue and abdominalwall skin.

FIG. 7. Comparison of the average 64-point variance in resis-tance in untreated (control) and 8-br-cAMP-treated (4 mM) fibro-blasts from TAO and normal orbital connective tissue and abdominalwall skin. Monolayers were allowed to proliferate to confluence be-fore any experimental manipulations were undertaken. Data werecollected 24 h after the shift to fresh medium without or with 8-br-cAMP.

365MORPHOLOGY CHANGES IN ORBITAL FIBROBLASTS

which became apparent at the light microscopic levelonly after several hours. Thus ECIS is a powerfulquantitative tool providing insight into the dynamics ofmorphological change associated with elevated cAMPlevels. When examined with ECIS, cells in culture ex-hibit rapid, random changes in resistance and capaci-tance. These changes are termed micromotion. Whencurrent flows from the small, cell-covered electrode inthe ECIS chamber, most of this current must flowunder the cells, in the narrow space between the ven-tral surface of the cell and the electrode, before escap-ing into the solution. The flow of current betweenneighboring cells is dependent in part on barrier func-tion. ECIS measurements are very sensitive to changesin the current path and this property can be used toexplore the effect of a wide variety of stimuli uponmicromotion. These studies provide insight into theapparent differences among fibroblast culture strainswith regard to micromotion. It would appear that or-bital fibroblasts may exhibit considerably more micro-motion than dermal cells. The average variance inresistance of cell micromotion was calculated usingperiods of 64 s [22]. Orbital fibroblasts have an averagevariance in resistance which is severalfold higher thanthat observed in dermal fibroblasts. The patterns ofmicromotion found in orbital fibroblasts from patientswith TAO and in normal orbital fibroblasts are distinct,as is reflected in different variance values. When or-bital fibroblasts are treated with compounds whichincrease cAMP levels, they behave more quietly inculture and their resistance values decrease. This ef-fect can be seen in the data displayed in Fig. 6. Incontrast, micromotion in dermal fibroblasts is in-creased with elevation of cAMP levels, as are the re-sistance values. Further studies are needed to definethe mechanism through which cAMP elicits morpho-logical changes in orbital fibroblasts and how this re-lates, on a molecular level, to the fibroblast type-spe-cific effects on cellular micromotion.

This work was supported in part by National Institutes of HealthGrants EY 08976 and EY 11708 (to T.J.S.), by the Research Serviceof the Veterans Affairs Medical Center (to T.J.S.), and by a contractfrom the National Foundation for Cancer Research.

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Received June 1, 1998Revised version received September 9, 1998

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