(CANCER RESEARCH 50, 4377-4381. July 15. 1990]

Cerebral Vasomotor Responses after Recombinant Interleukin 2 Infusion1

Mary D. Ellison, Richard J. Krieg, and Randall E. MerchantDepartment of Anatomy, Medical College of Virginia, Richmond, Virginia 23298-0709

ABSTRACT

The effects of systemic human recombinant interleukin 2 (rIL-2)infusion upon both the vasoconstrictor effect of hypocapnia and theendothelium-dependent vasodilator effect of acetylcholine (Ach) wereexamined in anesthetized rats equipped with cranial windows. Prior tothe functional studies, each of six animals received an i.v. infusion of rlL-2 (6 x 10* lU/kg) every 8 h for 3 days. At the same time, six control

animals received infusions of equivalent volumes of sterile water. Eighth after the final infusion, each animal was anesthetized and equippedwith a cranial window for the observation of pial arterioles overlying theleft frontoparietal cortex. Pial arteriolar diameters were measured beforeand after the topical application of Ach which in normal cerebral arterioles elicits the release of endothelium-dependent relaxing factor, causingvasodilation. When arteriolar diameters returned to base line, they weremeasured again both before and during hyperventilation-induced hypocapnia. Following functional assessments, these same pial vessels wereprocessed for study by transmission electron microscopy to determine ifany observed functional changes correlated with morphological abnormality.

Results of the statistical analyses suggested that normal Ach-inducedendothelium-dependent vasodilation was absent in the rIL-2-infusedgroup. Additionally, these animals exhibited reduced reactivity to thevasoconstrictive effects of arterial hypocapnia. The control group exhibited normal responsiveness to both Ach and hyperventilation. Ultrastructural studies revealed occasional morphological alterations of both vascular smooth muscle and endothelial cells in some vessels of rIL-2-infused animals but not in controls. These data suggest that repeatedsystemic rIL-2 infusion results in altered vasomotor responsivenesswithin the cerebral microcirculation. The data also suggest that theobserved vasomotor changes are not always accompanied by overt morphological alterations of either endothelial or smooth muscle cells.

INTRODUCTION

Immunotherapy involving high-dose i.v. administration ofrecombinant interleukin 2, with or without lymphokine-acti-vated killer cells, has shown some efficacy in mediating theregression of certain forms of metastatic cancer (1). Importantly, both laboratory and clinical studies have shown that rlL-22administration is accompanied by dose-limiting toxicity char

acterized, in large part, by a diffuse systemic capillary leaksyndrome of undetermined etiology (2, 3). This increase invascular permeability results in profound tissue edema, hypotension, and, in some cases, respiratory distress. In addition tothese side effects which are clearly related to vascular injury, anumber of patients also develop clinically significant neuropsychiatrie changes which appear to be dose related and, therefore,treatment limiting (4).

In order to investigate a potential link between such neuropsychiatrie changes and altered vascular permeability in thebrain, we initially examined blood-brain barrier status, in catsand rats, following i.v. rIL-2 infusion (5,6). The results of those

Received 11/20/89; revised 2/26/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this faci.

1This work was supported by Grant NS25871 from the NIH and the R. Clifton

Brooks, Jr.. Scholarship from the Sons of Confederate Veterans.2The abbreviations used are: rIL-2, recombinant interleukin 2; Ach, acetylcho

line hydrochloride; EDR. endothelium-dependent relaxation; EDRF. endothelium-dependent relaxation factor; cGMP. cyclic guanosine 3':5'-monophosphate;IL-2, interleukin 2; TNF, tumor necrosis factor.

studies revealed that single rIL-2 infusions, at clinically relevantdoses, induced alterations in cerebrovascular permeability toprotein. We therefore sought, in the present investigation, toextend our study of the cerebrovascular consequences of rIL-2infusion by examining two aspects of cerebral arteriolar vasomotor function following multiple i.v. infusions of rIL-2 in rats.In each animal, we examined whether rIL-2 had any effect onthe cerebral endothelium-dependent vasodilator response totopically applied 10~7 M Ach (Sigma, St. Louis, MO) (7-9).

We then examined, in the same vessels, effects on the normalvasoconstrictor response of cerebral microvessels exposed tolow levels of arterial carbon dioxide (10, 11).

Our findings indicated that, after 3 days of rIL-2 infusions,pial arterioles exhibited impairment of endothelium-dependentvasodilator mechanisms as well as reduced smooth muscle-mediated responsiveness to the vasoconstrictor influence ofarterial hypocapnia. These findings suggest that cerebral vasomotor function is impaired in rats administered multiple infusions of rIL-2. Such vasomotor deficits, if widespread, couldhave implications for the regional regulation of cerebral bloodflow (12).

MATERIALS AND METHODS

Animals. Sixteen adult male Sprague-Dawley rats, 250-325 g, wereused. Eight received i.v. injections of rIL-2 and 8 were infused withsterile water, the required diluent for Proleukin (see below). Prior todata analysis, two of the rIL-2-infused animals were excluded from thestudy because, under the influence of anesthesia, their mean arterialblood pressures had dropped below 65 mm Hg. One water-infusedanimal was lost due to ventilator failure while another died from anobstructed airway.

rIL-2. Recombinant human interleukin 2 (Proleukin) was kindlysupplied in sterile vials by the Cetus Corp. (Emeryville, CA). Each vialcontained 1.2 mg (18 x IO6lU/mg, equivalent to 3 x IO6Cetus units/mg) rIL-2, lyophilized, which was reconstituted prior to use with 1.2ml sterile water for injection. The dosage used in each infusion was 6x 10s lU/kg delivered into an indwelling cannula in the external jugular

vein. This dosage level was selected because it is commonly used inclinical trials (1). Control animals received equivalent volumes of sterilewater.

Experimental Design. Each animal was briefly anesthetized with etherwhile the right external jugular vein was cannulated orthograde. Thedistal end of the cannula was then passed s.c. to the posterior aspect ofthe neck where it was exteriorized and capped. Forty-eight h afterrecovery from anesthesia, each animal was infused with either rIL-2 orsterile water. Eight rats received a total of nine bolus rIL-2 infusions,delivered at 8-h intervals for 3 days. An additional eight animalsreceived sterile water infusions 3 times daily for 3 days. Eight hfollowing the final infusion, each animal was anesthetized with sodiumpentobarbital (40 mg/kg i.v.), tracheotomized, and equipped with afemoral arterial cannula for blood pressure and blood gas monitoring.Each animal was also fitted with a cranial window for visualization ofthe left frontoparietal pial microcirculation (see below). Followingsurgical manipulations, each animal was paralyzed (gallamine triethiod-ide, 4 mg/kg i.v.) and ventilated with room air supplemented by <).. asneeded. Body temperature was maintained at 37°Cwith the aid of a

heating pad. As described below, pial arteriolar vasomotor responseswere then examined after topical Ach application and hyperventilation-induced hypocarbia. After completion of the cranial window studies,each animal was administered a lethal dose of sodium pentobarbital

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CEREBRAL VASOMOTOR RESPONSES AFTER lL-2 INFUSION

and perfused transcardially with 0.9% sodium chloride followed byfixative consisting of 2% paraformaldehyde and 2.5% glutaraldehydein 0.1 M phosphate buffer. The morphology of pial vessels was studiedby transmission electron microscopy.

Vasomotor Assessments. Each animal was equipped with a cranialwindow modified from those used in cats (13) and rats (14). In preparation, the head was securely positioned in a rodent stereotaxic device.Following reflection of soft tissue, the left parietal bone was thinnedwith a razor blade and carefully removed without disturbing the underlying dura mater. With the aid of a dissection microscope, the dura wasreflected revealing the left frontoparietal pial microcirculation. Thecranial window, constructed of dental acrylic, bone wax, and a roundglass coverslip, was applied to the remaining bone with dental acrylic.The space under the window was then Tilledwith artificial cerebrospinalfluid (14) by way of inlet and outlet ports constructed of blunt-end 23-gauge needles embedded in the window wall. No components of thewindow were in contact with the brain or the dura. Vasomotor assessments were accomplished through the use of a Leitz/Wild stereomicro-scope fitted with a Vickers image-splitting device (13) and were initiatedwhen both arterial blood pressure and blood gas levels were withinnormal limits. Prior to the first vasomotor assessment, vessels in thefield were depicted in a sketch designating arterioles selected for study.In this manner, repeated measurements of the same vessels could beperformed. To initiate the assessment of endothelium-dependent relaxation, the diameters of the selected pial arterioles were measured forthe first time. A solution of 10~7M Ach in mock rodent cerebrospinal

fluid was then superfused under the window and the diameters of thesame arterioles were immediately measured again. No more than 10min after Ach application, the window was flushed with artificialcerebrospinal fluid. Fifteen min thereafter, just prior to hyperventila-tion, an arterial blood gas sample was drawn and base-line diametersof the same arterioles were again recorded. The ventilatory rate wassubsequently increased such that the arterial PaCOz level was reducedby 50% within 15 min. Vascular measurements were repeated to assessthe vasoconstrictor response to hypocarbia. The ventilatory rate wasthen reduced until arterial PaCO2 values returned to normal. Finally,in each rat, if any vessel had failed to dilate after Ach application, athird vasomotor challenge was conducted to determine whether theobserved impairment could be related to smooth muscle dysfunction.In that EDR requires not only the production of EDRF by endotheliumbut also the generation of cGMP by vascular smooth muscle (15), wewished to examine vascular responses to another agent which, likeEDRF, relaxes vessels by increasing smooth muscle cGMP but does soindependent of endothelial function. For this purpose, vascular responses were measured before and after topical application of sodiumnitroprusside (0.25 Mg/ml cerebrospinal fluid; Sigma) (16). Followingthe nitroprusside test, the animals were perfused.

Histological Preparation. Following brain removal from the calvaria,the cortex and pia underlying the window were dissected free from therest of the brain. Using the sketch of the pial vasculature beneath thewindow, the specific vessels studied functionally were identified (17),separated from the underlying cortex, and prepared for ultrastructuralanalysis. Pial arterioles were also sampled from the opposite hemisphere which had remained covered by bone throughout the experiment.

Data Analysis. The number of vessels studied functionally rangedfrom 6 to 16 arterioles/animal, depending on the number of vesselsvisible in the field. The total number of vessels examined was 71 inrIL-2-infused animals and 54 in controls. For each vessel, in eachanimal, the percentage of change in vascular diameter (percentage ofbase line) observed following Ach application was calculated. Then, foreach animal, a mean and standard deviation for the percentage ofchange in diameter exhibited by all of the measured arterioles of thatanimal was determined. The means for the percentage of diameterchanges exhibited by rIL-2-infused animals were then averaged and,using standard deviations derived for each animal, a pooled standarddeviation and a standard error for the group were also calculated. Thevasomotor data gathered following Ach application in control animalswere then treated in the same manner. The mean Ach-induced responseof the rIL-2-infused group was compared to the responses of the water-infused group by a 2-sample / test.

The same procedure was followed in determining whether vasomotorresponses to hyperventilation-induced hypocapnia differed between rlL-2-infused and control animals.

Additionally, the morphology of 12 vessels/group, from controls andrIL-2-infused animals, was examined in relation to the corresponding

functional analyses.

RESULTS

Following femoral artery cannulation, mean arterial bloodpressure levels for both animal groups were within normallimits throughout the surgical procedures and vasomotor assessments (Table 1). Through adjustments in ventilatory rateand stroke volume, arterial blood gas values were maintainedwithin normal limits, except during hyperventilation (Table 1).Diameters of the vessels selected for vasomotor assessmentsranged from 40 to 101 /¿mbefore Ach application.

In control animals, the normal vasodilator response of cerebral arterioles to topical Ach (9) was observed (Table 1; Fig. 1).Arteriolar diameters increased by 11 ±2.8% (SE) of base line.Similarly, hyperventilation induced the normal cerebral vasoconstrictor response (10, 11) to arterial hypocapnia, reducingarteriolar diameter by 12 ±2.6% of base line (Table 1; Fig. 1).

Recombinant IL-2-infused rats showed altered vasomotor

responses to both topically applied Ach and hypocapnia. Individually, cerebral vessels of rIL-2-treated rats showed consid

erable variation in their responses to Ach; only 7 of 70 vesselsstudied exhibited normal Ach-induced vasodilation. Other vessels either dilated minimally, did not change their diameter, orconstricted. The calculated mean response of the 70 vesselsstudied following Ach exposure was a decrease in pial arteriolardiameter by 0.008 ±2.3%, effectively zero (Table 1; Fig. 1).This response differed significantly from that observed in thecontrol group (P < 0.01). After hyperventilation, the degree ofvasoconstriction induced by arterial hypocapnia was also significantly reduced (P< 0.025) as compared to the control group(Table 1; Fig. 1). Following topical application of sodiumnitroprusside, every vessel exhibited the normal vasodilatorresponse, suggesting that the observed impairment of Ach-induced EDR was not due to an impaired ability of smoothmuscle to generate cGMP.

Ultrastructural examination of pial vessels studied functionally revealed that the majority of vessels from both control andrIL-2-infused animals were morphologically normal, regardless

of their responses to Ach or hyperventilation. This observationis consistent with findings of others who have shown loss ofEDRF in the absence of ultrastructural change (18). Severalpial arterioles from rIL-2-infused animals, however, did exhibit

some endothelial cell and/or smooth muscle alterations (Fig.2). In these vessels, endothelial cells with increased cytoplasmiclucency were occasionally observed, interspersed among morphologically normal endothelia. These cells were sometimesseen in close proximity to smooth muscle cells which alsoexhibited morphological abnormalities in the form of largeelectron-lucent cytoplasmic inclusions. Such alterations of cer-

ebrovascular ultrastructure were reminiscent of those previouslyreported in studies of rIL-2-induced blood-brain barrier dysfunction (5, 6). Vessels harvested from the contralateral hemisphere, protected by bone throughout the experiment, showeda similar spectrum of morphological changes. Pial vessels fromcontrol animals exhibited no morphological abnormalities ofeither endothelial or smooth muscle cells.

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CEREBRAL VASOMOTOR RESPONSES AFTER IL-2 INFUSION

Table 1 Effect ofrIL-2 on pial arterialesAll values are means ±SEM. Responses were studied in 71 arterioles of 6 rIL-2-infused rats and in 54 arterioles of 7 control rats. Statistical comparisons of

responses to Ach and hypocapnia were compared between the two groups by using r tests.

Ach application Hyperventilation

Before(base line,) After

Before(base lim1..) After

Vessel diameter (jim) 68 ±2.5A diameter"

Due to Ach, % of base-line, valueDue to hyperventilation, % of base-line2 value

MABP (mm Hg) 105 ±5.8PaCO2 (mm Hg) 36 ±2.5PaO;(mmHg) 100 ±16.5pH 7.37 ±0.02

A. Control group

75 ±2.6 64 ±2.8

11 ±2.8

NANANANA

99 ±11.440 ±2.2

104 ±15.17.37 ±0.02

B. rIL-2-infused group

56 ±2.7

-12 ±2.6

117±5.121 ±1.1

120± 16.27.51 ±0.02

Vessel diameter(¿im)AdiameterDue

to Ach, % of base-line,valueDueto hyperventilation, % of base-line2valueMABP(mmHg)PaCO2(mmHg)PaO2

(mmHg)pH71

±1.990

±4.337±2.41I7±

17.07.38±0.0271

±1.90

±2.3*NANANANA70

±1.889

±3.536±2.3113±

17.97.39+ 0.0168.0

±1.8-3

±2.4C104

±19.019±0.5125

±12.97.51±0.03

" A diameter, change in diameter; MABP, mean arterial blood pressure; PaCO2, carbon dioxide tension; PaO2, oxygen tension; NA, not assessed.* Statistically significant as compared to Ach response in control group (P < 0.01).' Statistically significant as compared to hyperventilation response in control group (P < 0.025).

Ach Application

Ach

Hyperventilation

Hypocapnia [

3«

a.

40

20

-20Control rlL-2 Control rlL-2

Fig. 1. Changes in pial arteriolar diameter, expressed as percentage of baseline, following topical Ach and hypocapnia. Two groups of rats are shown. Controlanimals received water infusions and rIL-2 animals received IL-2. Columns, mean;bars, SE. The vasodilator response to Ach was significantly reduced in rIL-2-infused animals (P < 0.01) as was the vasoconstrictor response to hypocapnia (P< 0.025).

DISCUSSION

In summary, the results of the present study suggest that,secondary to multiple rIL-2 infusions in rats, there is impairment of cerebral vasomotor responses to physiological andexperimental stimuli. A number of previous clinical and laboratory studies have similarly reported IL-2-related vasculareffects including permeability increases, alterations of endothe-lial morphology and metabolism, and endothelial lysis (1, 2,19-21). In some cases, these effects appeared to be directlyattributable to IL-2 (19, 20), whereas, in others, host lymphoidcells have been implicated in IL-2-related vascular effects (21).This latter observation is supplemented by clinical and laboratory reports that rIL-2 can elicit the production of other solubleimmune factors with vasoactive properties (22-28).

If cerebral vessels, like their peripheral counterparts, can beinfluenced by cytokines, as the present findings suggest, theconsequences may have special significance for central nervoussystem function. Cerebral microvessels are equipped with special adaptations which strictly regulate not only blood-to-brainpassage of circulating solutes but also regional cerebral bloodflow. These adaptations exist for the purpose of maintainingoptimal metabolic conditions within the neuronal/glial mi-

Fig. 2. A, transmission electron micrograph revealing normal ultrastructuraldetail in pial arteriole from rIL-2-infused rat. Note that both the endothelium(/•.')and vascular smooth muscle (.SU) appear unremarkable. It. pial arteriole

exhibiting morphological alterations occasionally observed in rIL-2-infused animals but not in controls. Note that the vascular smooth muscle (SM) appearsnormal, exhibiting cytoplasmic and nuclear detail comparable to that shown inA. The adjacent endothelial cell, however, demonstrates abnormally lucent cytoplasm (arrows).

croenvironment. When conditions are such that these featuresare altered, the potential exists for adverse impact on neuronalfunction.

Among cerebral microvascular specializations is the capacityof small resistance vessels to change caliber in response to

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CEREBRAL VASOMOTOR RESPONSES AFTER IL-2 INFUSION

variations in systemic blood pressure or arterial content of O2and/or CO2 (10, 29). Another potential participant in theregional regulation of cerebral blood flow is an endothelium-derived vasodilator the release of which can be experimentallyelicited in vivo by topical 10~7 M Ach application (7). Impair

ment of both endothelium-dependent and -independent cerebralvasomotor responses has been demonstrated in a variety ofpathological and experimental conditions (9, 11). In that, recently, cytokine exposure has also been shown impair vasomotor responses (30), it seemed especially timely and appropriateto undertake the present study.

As to potential mechanisms underlying the presently described vasomotor dysfunction, there is a variety of possibilities.One explanation involves the potential role of free oxygenradicals, well known to be mediators of cellular injury andvasomotor dysfunction (9, 11,31). Support for this idea lies inreports by others that cytokines can enhance endothelial cellprostaglandin synthesis (32, 33), a metabolic process duringwhich free radicals are generated (34). Additionally, cytokineshave been shown to enhance adhesion between endothelial cellsand leukocytes (35), well known to induce vascular damagethrough free radical mechanisms (36). Furthermore, there isevidence that rIL-2-inducible cytokines can elicit Superoxideaniónrelease from endothelial cells themselves (37).

An additional explanation for the observed vasomotor dysfunction involves the potential role of TNF which we haveobserved to circulate at elevated levels in rats infused with rlL-2.3 In that the anti-EDRF effect of TNF has been reported in

feline cerebral arteries in vitro (30), it appears feasible thatcirculating TNF may play a role in the presently described invivo cerebral vasomotor deficits.

Of potential interest, an early animal study of rIL-2-relatedtoxicity reported that a component of the rIL-2 vehicle wasfound to contribute to rIL-2-induced histopathology and elevated serum liver enzymes (38). The vehicle formulation wassubsequently modified to reduce the concentration of the component, and, as such, there have been no recent reports ofvehicle-related toxicity. Comprehensive studies in our laboratory have, similarly, shown no alterations of cerebrovascularultrastructure or permeability (6) and no circulating TNF3

following vehicle infusion in rats. We are, therefore, confidentthat vehicle effects do not underlie the presently reportedvasomotor deficits.

In that pial arterioles contribute significantly to total cerebrovascular resistance (12), the observed dysfunction may haveimplications for cerebral blood flow in rats, and experiments toinvestigate this possibility are currently in progress. It may bemore interesting, however, to speculate as to the potentialclinical significance of the present findings. Patients undergoinghigh-dose rIL-2 treatment commonly develop a variety ofhemodynamic changes including hypotension and decreasedsystemic vascular resistance (1, 39). It is feasible that suchchanges, compounded by cerebral vasomotor deficits, couldpredispose the brain to suboptimal blood flow levels in somebrain regions (40), with potential metabolic consequences.

ACKNOWLEDGMENTS

We wish to thank Mary Lee Giebel and Susan Walker for theirtechnical assistance and DeJun James and Elaine Pollard for their helpin the preparation of this manuscript.

J Unpublished observations.

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1990;50:4377-4381. Cancer Res   Mary D. Ellison, Richard J. Krieg and Randall E. Merchant  InfusionCerebral Vasomotor Responses after Recombinant Interleukin 2

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