0022-202X/82/7805-0386$02.00/0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 78:386-390, 1982 Copyright © 1982 by The Williams & Wilkins Co.

Vol. 78, No.5 Print ed in U.S.A.

The Effect of Epicutaneous Glucocorticosteroids on Human Monocyte and Neutrophil Migration in Vivo

DAVID A. NORRIS, M.D., LESLIE CAPIN, B.A., WILLIAM L. WESTON, M.D.

Department of Dermatology, University of Colorado School of Medicine, Denver, Colorado, U.S.A.

The effect of epicutaneous methyl prednisolone (MP) at 10-\ 10-5

, and 10-6 molar concentration was studied in 54 normal, healthy volunteers using a new, in vivo microchemotaxis technique. Significant inhibition of monocyte chemotaxis occurred at all concentrations studied and persisted over a 24-hr period with 10-4 molar MP. Neutrophil chemotaxis was significantly inhibited only with 10-4 MP. The inhibitory effect ofMP on neutro­phil and monocyte chemotaxis occurred earlier and at lower concentrations if the skin sites were pretreated with steroid. Thus, when corticosteroids are applied on , abraded skin in concentrations achievable in vivo, mono­cyte chemotaxis into tissue is inhibited for longer periods and at lower drug concentrations than is neutrophil chemotaxis. By avoiding the significant systemic effects of corticosteroids on circulating monocyte and neutro­phil populations, these experiments establish that local inhibition of chemotaxis is an important anti-inflamma­tory effect of corticosteroids, with differential effect on monocytes and neutrophils.

During 20 years of clinical use, the efficacy of systemic and local glucorticosteroids has been clearly demonstrated in the treatment of immunologic and inflammatory disease, but the major site(s) of action of steroids provide a complex combina­tion of immunosuppressive and anti-inflammatory effects. Corticosteroids are effective inhibitors of specific T lymphocyte functions. In man, the ability of T lymphocytes to respond to antigenic and mitogenic stimuli with a blastogenic response and lymphokine release is diminished with long-term daily steroid treatment, [1-5] but these effects may be due largely to the profound redistribution of the recirculating small lymphocytes, [6-10] produced rapidly by even alternate-day steroids. B cell function as manifested by antibody production is inhibited in. vitro and in. vivo by steroids, [11,12] and the ability of specific antibody to induce tissue inflammation is also inhibited by steroids. [13]

In addition to these specific immunosuppressive effects of steroids, numerous significant anti-inflammatory effects of steroids alter the availability of nonspecific effector cells at tissue sites of inflammation and block the translation of specific immunologic signals into inflammatory responses. At clinically relevant doses, on alternate day regimens steroids produce a rapid but transient monocytopenia, lymphopenia, eosinopenia,

Manuscript received May 22, 1981~ accepted for publication October 26, 1981.

This work was supported by grants from the ' Upjohn Company and the National Cancer Institute, 5 R01 CA26760-05, NIH.

Presented at the P lenary Session of the Western Section of the American Federation for Clinical Research and the Western Society for Clinical Research, February 7, 1980, and at the National Student Research Forum, April 23, 1981.

Reprint requests to: David A. Norris, M.D. , Department of Derma­tology/Box B153, University of Colorado School of Medicine, Denver, CO 80262. .

Abbreviations: HBSS: Hanks Balanced Salt Solu tion MP: methyl prednisolone ZAS: zymosan activated autologous serum

and a neutrophilia. [8-14] This is associated with a decreased availability of monocytes at sites of tissue inflammation [15-18] and in some instances a paradoxical decrease in neutrophil availability in tissue secondary to a decrease in the mluginal pool of neutrophils associated with diminished adherence of neutrophils to endothelial cells. [20-24] In contrast to the pro­found changes in cell distribution and availability induced by systemic steroids, direct inhibition of monocyte and neutrophil function is difficult to achieve at phannacological concentra­tions of steroids. [24-27]

In evaluating the anti-inflammatory effect of steroids on tissue inflammation, it has been difficult to separate the sys­temic effects on cell populations from local effects on the migration of effector cells into tissue. Previous studies of tissue applications of steroids have been either semiquantitative, have failed to accurately differentiate neutrophil and monocyte mi­gration, have used nonpharmacological levels of drug or have used assays with inappropriate nonphysiological barriers to cell migration. [28-30] In the study presented here we have used a new in. vivo microchemotaxis technique to quantitatively test the effect oflocal steroids in pharmacological concentrations on monocyte and neutrophil migration toward complement-de­rived chemotractants without the interfering influence of ste­roids on peripheral blood cellular populations. We have dem­onstrated that local inhibition of chemotaxis of monocytes and neutrophils in human tissue in. vivo is a significant anti-inflam­matory effect of steroids. Monocytes are clearly more suscep­tible to this effect than are neutrophils; the effect is dose and time dependent, and is achieved at steroid concentrations read­ily obtained in. vivo.

METHODS

Subjects

Various aspects of the effects of epicutaneous corticosteroids were studied in 54 normal human volunteers. Subjects served as their own controls. Persons with recent illness, ch.ronic skin disease, or who were taking medications were exclucl p.rl.

Chemotaxis Technique

Details of this quantitative in vivo neutrophil and monocyte che­motaxis technique have been described previously. [31] Betadine and alcohol-sterilized skin was tape abraded with Blenderm tape (3M Co., St. Paul, MN) over 5-mm templates to produce 6 uniform glistening abrasions without macroscopic bleeding. Small plexiglass chambers of .2 ml volume containing 5U% zymosan activated autologous serum (ZAS) [32] diluted in Hanks Balanced Salt Solution (HBSS) were recovered with a Nucleopore filter sandwich (Nucleopore, Pleasanton, CAl composed of a 3 /l and a 5 /l pore filter and inverted over the abrasion. In this way, cells migrating from the abrasion toward the attractant were required to pass first through the 5 /l pore filter and then through the 3 /l pore filter. Monocytes were relatively relarded within the filter sandwich, while neutrophils passed more freely through the 3 /l pore ftlter. Chambers were kept in place for various periods and removed for quantitation. To determine the total number of cells which migrated, the 2 filter sandwich was removed and treated as follows: the attractant side of the 3}J. filter was scraped until glistening to remove the mass of neutrophils accumulated there, and the number of cells in t he scrapings were counted. The filter sandwich was separated and stained for monocyte esterase activity (alpha napthyl acetate esterase) [31,32] and mounted for counting. This stain a llows accurate differen­tiation of monocytes, neutrophils and lymphocytes. Differential counts of monocytes and neutrophils were performed on each filter, and the

386

May 1982 LOCAL GLUCOCORTICOID EFFECT ON CELLULAR CHEMOTAXIS IN VIVO 387

total number of each type of cell in 10 random fields was multiplied by a conversion factor to give the total number of cells per 5-mm abrasion. The total numbers of monocytes and neutrophils in the resuspended scraping and on the ftlters were added to obtain total monocyte and neutrophil migration. The relative numbers of neutrophils in the scrap­ing versus those within the ftlters were not altered by steroids, nor was the relative percentage of monocytes which crossed to the 5 f.L and 3 f.L filter. With the filter combination chosen, and at the time studied, greater than 50% of the cells which did not cross the 3 f.L ftlter were monocytes, and fewer than 5% of the cells which did cross the ftlter were monocytes. Using this technique, lymphocytes were not shown to migrate in response to complement-derived chemotractants.

Experimental Design

In these experiments, subjects were used as their own controls by dividing 6 tape abrasions into 3 pairs. One member of each pair was covered with a chamber containing steroids or one containing HBSS diluent control. After the prescribed preincubation period, the chamber was removed and replaced with an attractant-containing chamber for the assay of subsequent monocyte and neutrophil migration . In this way the effect of varying steroid concentrations and periods of prein­cubation of steroids on abraded skin was studied over different inter­vals. Each data pair was analyzed independently and the significance of the effect of each variable was determined using a paired t-test. In this way, individual or anatomical variations in response were con­trolled.

The steroid used in these experiments was methyl prednisolone sodium succinate (SoluMedrlo, Upjohn Co., Kalamazoo, MI) , diluted in HBSS to concentrations that have been shown to be physiologic (IO- OM), pharmacological (lO-OM), and suprapharmacological (10- 'M). [34) The suprapharmacological concentration was included because it has not been clearly shown whether a concentration of corticosteroids at 10- 4 Molar might be achieved locally in vivo with application of clinically availab le topical preparations (l0- 3M) of corticosteroids.

Subjects were grouped and such variables as steroid concentration, duration of preincubation, and duration of inhibition were studied, using each subject as his own control. Data were compiled both as absolute cell migration with and without steroids and also as the inhibition of migration in thousands of cells, which represents the numerical difference between each experimental chamber and its paired diluent control.

CHEMOTAXIS: 10-4 MOLAR MP

60 Monocyte

50 o = Control

'( 40

Cl = Steroid 0

< .01 < .05

n=9

X 30

en 20

-' LLJ U

0 Neutrophi I LLJ

t--< "" <.::>

.,.01

~ 300

200

100

4 8 12 HOURS

FIG 1. Effect of 10-" molar methyl prednisolone (MP) on monocyte and neutrophil migration at 4, 8, and 12 hr. The accumulation of cells in response to ZAS is shown after 4, 8, and 12 hr collections. Differential counts of monocytes and neutrophils are shown for abrasions which had been treated with epicutaneous MP or with HBSS control. The bars indicate mean cell migration in thousands ± standard error of the mean (SEM). Statistical significance-indicated above each pair of bars-was determined by paired t-testing of steroid and HBSS-pre-treated chamber pairs. .

CHEMOTAXIS : 10-5 MOLAR MP

Monoe y te

50 D =Control

'? 40 m =Steroid

0 n =6 p = .02

30 p =.001 x Vl 20 -' w

10 NS U

0 w Neutrophi I >-<:

"" ~ 200

~ NS

p= .05

4 8 12 HOURS

FIG 2. Effect of 10- 5 molar MP on monocyte and neutrophil migra­tion at 4, 8, and 12 hr.

CHEMOTAXIS: 10-6 MOLAR MP

60 Monocyte p=.OO';

50 [] = Control

'? 40 m = Steroid

0 n =11

X 30

Vl 20

UJ 10 U NS

0 Neutrop hil UJ

>-<:

"" t:> NS

~ 200

4 8 12 HOURS

FIG 3. Effect of 10- 0 molar MP .on monocyte and neutrophil migra­tion at 4, 8, and 12 hr.

RESULTS

The effect of preincubation of tape-abraded skin for 2 hI' with steroids is shown on cell migration at 4,8, and 12 hr in Fig 1-3 for groups of subjects in which methyl prednisolone (MP) at 10- 4

, 10- 5, and 10- 6 Molar concentration was used. In Fig 1,

significant inhibition by 10-" MP of both monocyte and neutro­phil migration toward ZAS was seen at all times assayed. At 1O- 5M MP, a concentration obtained with clinical systemic doses of steroids (Fig 2), both monocyte and neutrophil migration were still inhibited, but the duration and magnitude of inhibi­tion were clearly greater for monocytes than for neutrophils. At 10- 6 M MP (Fig 3), inhibition of chemotaxis was seen only with monocytes in experiments in which cells were collected for 12 hours.

Figure 4 shows the monocyte and neutrophil migration after 24 hours in a group of subjects in which MP at 10- \ 10- 5

, and

388 NORRIS, CAPIN, AND WESTON

1O- 6M concentration or HBSS controls were applied to abra­sions 2 hr before application of attractant. Although there is inhibition in numbers of monocytes accumulated over that period, with all MP concentrations the result is significant only with lO- "M MP. With neutrophils, there was evident decrease in mean migration, but comparison to pail'ed controls shows no significant inhibition. Close analysis of individual data pail's shows that with 1O-6M MP and with concentrations of steroids at 24 hr, inhibition of neutrophil migration was highly variable from individual to individual. In other words, while neutrophil migration was inhibited in some individuals, in others it was not, suggesting an individual variability in susceptibility to low concentrations of steroids and individual variability in duration of effect at the lower limits of effective concentration.

If one examines the effect of corticosteroids at different concentrations as the absolute decrease in thousands of migrat­ing cells, it is clear that significant inhibition by steroids is clearly both concentration dependent and relatively cell type

M

~ 20

x V"l -' -' UJ

o

a UJ

>­<{ 0<

10

o 200

~

CHEMOTAX IS: 24 HOU RS Monocyte

NS NS

Neutrophil

NS

control 10 -6 10-5

n=6 o =Contro l

[] = Steroid

10-4 Mo lor MP

FIG 4. Effect of MP on monocyte and neutrophil migration over 24 hr. Migrating cells (in thousands) were collected over 24 Ill". Open bars are composite controls and shaded bars are the mean migration from abrasion sites pretreated for 2 hr with either 10- 4

, 10- ", or 10-" molar MP. Evaluation of data with a paired t-test inclicated that the progres­sive decrease in migration at higher MP concentrations was not sig­nificant (NS) except for monocytes at 10- 4 molar MP.

Vol. 78, No.5

specific. Inhibition of monocyte chemotaxis in vivo by local MP occurs at lower concentrations and persists longer at higher MP concentrations than does inhibition of neutrophil chemotaxis. Although the magnitude of inhibition of neutrophil migration in absolute numbers is far greater than the inhibition of mono­cyte migration, the relative percent inhibition is comparable between monocytes and neutrophils at concentrations where they are both susceptible.

The importance of pretreatment with MP is ad(iTessed in the Table. The inhibition in thousands of cells per chamber by MP at 10- 4 and 10- " concentration is expressed for chambers in which the MP was added with chemotractant or in which the abrasion site was treated with MP before application of chem­otractant. Although significant inhibition of monocyte chemo­taxis was achieved with 10-" M MP with or without pretreat­ment, pretreatment produced an earlier onset of effect. Inhibi­tion of monocyte migration with 10- 5

M MP is also delayed in onset and never reaches significant levels without pretreatment of the tissue before application of atractant. The absolute magnitude of inhibition of neutrophil chemotaxis is even more significantly diminished by adding steroid to attractant without pretreatment, and again the onset of inhibition is delayed . From this experiment, however, one cannot be sure whether the delay in onset of effect or the limitation in magnitude of steroid­induced inhibition of cell accumulation is due to direct interfer­ence of activated serum with steroid penetration or to an absolute requirement of pretreatment of migrating cells and skin with steroids for maximal effect.

DISCUSSION The demonstration of a dil"ect effect of epicutaneous corti­

costeroids on monocyte and neutrophil migration in vivo dem­onstrates an imporant effect of steroids not clearly defined by previous in vivo and in vitro experiments. The technique used here to identify the local effect of steroids on cell ular migration provides several important advantages over the Rebuck Skin Window or other skin chamber techniques used previously to study cellular migration in vivo. This technique provides a physiological barrier to migration, a reservoil' of a known chem­otractant, and quantitative differential counts of monocytes and neutrophils studied at different times. The barrier to mi­gration is anatomically intact: tape stripping removes stratum corneum and some cells of the stratum granulosum. [35] Cells migrating into the chemotaxis chamber must pass through the postcapillary venules and the papillary dermis, through the basement membrane of the derma to-epidermal junction, and through an intact epidermis before accumulating within the Nucleopore filter sandwich. The attractant is autologous acti­vated serum complement, without which only a weak neutro­philic infiltrate develops. In the Rebuck technique, the abrasion of skin often removes most of the epidermis and itself induces

Effect of 2 hr preincubation of methyl prednisolone on chemotaxis: !:J.M in. thousands (mean ± SEM)

Pretreatment

Monocytes 2 hr

o hr

:2 hr

o hr

Neutrophils 2 hr

o hr

2hr

o hr

" Compal'ed with paired medium control.

MP Concentration

lO- "M (n=6)

lO- f'M (n=8)

lO- "M (n=6)

lO- r'M (n=8)

4 hr

6.0 ± 2.1 p < .05"

1.7 ± 4.3

4.9 ± 2.9

1.4 ± 1.0

80.3 ± 19 p < .01

6.4 ± 7.1

31 ± 15.4

6.2 ± 9

8 hI'

14.0 ± 8.0 p < .05

20.7 ± 7.6 p < .05

12 ± 1.6 p < .001

10.9 ± 6

107 ± 26 p < .01

27 ± 10 P < .05

32 ± 29

44 ± 23

12 hI"

18.4 ± 5.0 p < .0]

15.8 ± 6.1 p < .05

25.8 ± 6.7 p < .01

3.9 ± 4.6

168 ± 34 p < .01

27 ± 8 p < .01

20 ± 69.9

69 ± 45

May 1982 LOCAL GLUCOCORTICOID EFFECT ON CELLULAR CHEMOTAXIS IN VIVO 389

the migration of inflammatory cells even without the addition of exogenous attractants. Quantitation in the Rebuck Window technique requires migrated cells to attach to a glass coverslip applied to sites of abrasion. Since steroids affect cell adhesion, the decrease in numbers of cells counted with the Rebuck Window may merely represent a decrease in the ability of cells to attach to glass surfaces, rather than diminished migration. Conversely, in most skin chamber techniques, the cells are counted in the chamber fluid after detaching from the abraded surface through which they migrate. Steroid-induced detach­ment from the abraded surface might cause the inhibitory effects of steroids on cell migration to be underestimated. In our microchemotaxis chamber technique, cells are counted as they migrate through a filter trap placed between attractant and abrasion-thus decreasing the effects on quantitation of steroid-induced reduction in cell adhesiveness. The use of uni­form reservoirs for application of known steroid concentration in ow' technique allows the induction of significant local effect without the necessity of systemic administration of steroids.

Such deficiencies in technique are reflected in previous re­ports of the effects of steroids on cellular migration in vivo. Rebuck and Mellinger [29] reported significant inhibition of cellular infiltrates by local steroids, but the concentrations used were apparently suprapharmacological, the barrier to migration was limited, and quantitation was semi-quantitative. Peters et al [30] reported that the absolute numbers of leukocytes accu­mulating in skin chambers in response to clotted serum actually increased with dexamethasone and decreased somewhat with prednisone; however, leukocyte clearance (migrated cells/ pe­ripheral blood WBC) was diminished in both, reflecting the profound neutrophilia produced by both steroid preparations. In that study, differential migration of monocytes and neutro­phils was not measured, the effect of steroids on the systemic availability of cells was misleading, and the measured end­point-cells in suspension in the skin chamber after detachment from the abrasion-may have been differentially affected by such steroids with known differences in glucocorticoid potency.

Several in vivo techniques have shown that systemic steroids ha ve a profound effect on monocyte availability at sites of tissue inflammation [k5,18] but again, it is not clear whether the effect on cellular availability was systemic or local. The most success­ful attempt to addJ'ess the effect of steroids on monocyte and neutrophil chemotaxis in vivo was the investigation by Dale, Fauci, and Wolff of the Rebuck Skin Window response of 20 patients with systemic inflammatory disease treated with daily and alternate day steroid therapy. [19] They found profound monocytopenia and decreased monocyte chemotaxis in patients on daily steroids on the "Day On" alternate day steroids. Neutrophilia and an elevated circulating neutrophil half-life was produced by daily steroids on the "Day On" alternate day steroids, but neutrophil chemotaxis in vivo was only depressed on daily steroids. Monocyte and neutrophil numbers and func­tions were normal on the "Day Off' alternate day steroids. They established both a differential effect of steroids on mono­cyte and neutrophil availability in tissue inflammation, and the apparent safety of alternate day steroid regimens in allowing effector cells to be available at sites of inflammation. However, the obligatory use of patients with significant immunologic disease as subjects likely selected a population with altered reactivity, as evidenced by the rather large differences in mono­cyte chemotaxis in controls and patients on the "Day Off' steroids. Also, although the marginal pool of neutl'ophils was apparently decreased on the "Day On" alternate day steroids, neutrophil chemotaxis was normal. While monocyte accumu­lation as judged by a weighted response over 24 ill' was normal on the "Day Off' steroids, migration between 12 and 24 h1' appeared to decrease for unexplained reasons. In the final analysis this classic study did not clea1'ly define the differences between local and systemic effects of steroids on monocyte and neutrophil migration in vivo, and encompassed the shortcom­ings of the Rebuck Window technique: non physiological bar-

riel'S', poor attractant specificity, inadequate differentiation of monocytes and neutrophils, and dependence on cell adherance for counting and a semiquantitative endpoint. In contrast, the experiment which we present uses normal subjects who serve as their own controls, measures local and not systemic effects of corticosteroids, more clearly defines tissue dose response, provides quantitative specific differentiation of monocytes and neutrophils, and provides a timed picture of both induction and duration of inhibition.

Examination of the effects of steroids on monocyte function in vitro has produced a confusing mosaic of positive and nega­tive effects. Rhinehart et al. have shown that suprapharmaco­logical concentrations of steroids in vitro inhibit monocyte chemotaxis, random migration, and staph bactericidal activity but do not affect neutrophil chemotaxis or monocyte crypto­coccal phagocytosis, IgG receptor function or glass adherence. [36] On the other hand, when normal healthy subjects were fed significant clinical doses of steroids (prednisone 50 mg po q12h X 6), chemotaxis, cryptococcal phagocytosis, hexose monophos­phate shunt activity, and ultrastructural features of harvested peripheral blood monocytes were not altered [37], only Staph Aureus and Candida tropicalis killing were depressed. It has also been shown [14] in patients receiving clinically relevant doses of alternate day steroids that chemotaxis, phagocytosis, and bacterial killing by periphel'81 blood monocytes were nor­mal although profound monocytopenia occurred even at low doses of steroids. Other investigators have also shown variable effects on phagocytosis and killing of different organisms [38] and on monocyte maturation into macrophages [33] by steroids. Thus while it is easy to decrease the peripheral availability of monocytes with pharmacological doses of steroids, overt inhi­bition of isolated in vitro monocyte functions , is difficult. A crucial problem in many experiments studying the effects of steroids on monocyte function in vitro is how to present the drug with appropriate carrier to potentiate cell absorption; [12] if this problem is not overcome, the levels of dJ'ug required to achieve effect in vitro are often much greater than those which in vivo will produce the same degree of effect [40]. Comparison of the difficulties in inhibitory monocyte chemotaxis in vitro with the same results presented here underscore this problem. We have demonstrated' that steroids presented locally in vivo in the millieu of serum and extracellular proteins clearly inhibit monocyte migration at pharmacological levels without any effect on the total monocyte pool available to tissue. In addition, this chemotaxis technique-which measures the ability of cells to cross vascular endothelium, the derma to-epidermal junction, and the epidermis-is a more critical and sensitive means of judging the total chemotactic response of monocytes than is measuring the cells' ability to cross or enter a thin filter in response to attractant. However, the effects attributed to ste­roids by these experiments may not be due to alteration of the migratory machinery of inflammatory cells but may instead affect local margination or the cells' ability to cross endothe­lium, ' dermato-epidermal junction or epidermis.

The local effect of steroids on neutrophil availability in tissue is clearly less profound than the effect on monocytes. In vitro inhibition of neutrophil chemotaxis is achieved only at supra­pharmacological levels [41,43]. At pharmacological levels, neu­trophil adherance [24] but not chemotaxis [26,27] or phagocy­tosis [43] are affected.

The standard technique for evaluation of the potency of topically applied steroids is the vasoconstrictor assay of Mc­Kenzie and Stoughton [44] which utilizes graded degrees of vasoconstriction to evaluate corticosteroid potency. The dem­onstration of differential effects of steroids on monocyte and neutrophil accumulation in microchemotaxis chambers strongly suggests that vasoconstriction alone is not necessarily a repre­sentative measure of the anti-inflammatory effects of steroids and further techniques for evaluating steroid potencies in vivo must consider the inlportant biological differences in monocyte and neutrophil susceptibility documented here.

390 NORRIS, CAPIN, AND WESTON

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