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32 Jensen LX Jorgensen JOL. Risteli J, Christiansen JS, Lorenzen I. Type I and III procollagen propeptides in growth hormonedeticient patients: effects of increasing doses of GH. Acta Endocrinol (Copenh) 199 1:124:27X-82. 33. Wolthers OD. Pedersen S. Growth of asthmatic children during treatment with budesonide: a double blind trial. BMJ:203: 163 5. 34 Schmid C, Guler HP, Roew D, Froesch ER. Insulin-like growth factor I regulates type I procollagen messenger ribonucleic acid

on bone r~?etabolism

Steady state levels in hone ot i.at\ II!!..lcvrmc~ii~~v 1989;125: 1575-x0. 35. Tut-p&en M. Sorva R, Juntunen-Backman I*;. i:!rsttgr~ in carbohydrate and lipid metabolism in asthmatic :hildretr inhaling budesonide. J AL.LBRGYCLIN IM~~~NOI, i3’+1:8X 3X1-9. 36. Risteli J. Niemi S. Elomaa 1. Bone resorption odium thiosulfate, washed in water, and stained with i:s.i)i toiutdinc blue, pH 7.4 for 3 minutes, dehydrated. and nounted in Permount (Fisher Scientific Co., Fair Lawn. ‘s4.J ! under glass coverslips. lmmunohistochemistry. At 2, 4, and 8 hours after intravenous antigen injection, 6 mm punch biopsies of PCA skin sites were fixed in 4% paraformaldehyde for 4 hours. dehydrated in graded alcohol solutions. and =rnbedded in paraffin. Two to 5 pm tissue sections were piaccd on glass slides. Specimens were heated at 56” C for i!) minutes, washed in xylene twice, rehydrated through graded alcohol solutions, and rinsed with distilled water. Endogcnous pcroxidase activity was exhausted by flooding each slide with 5% hydrogen peroxide in methanol for 5 minutes, Spccimens were washed twice with PBS with 0.01~~~BSA. All subsequent reagents were diluted in, and wash:? performed with 1% PBS with 0.01% BSA. lmmunoperoxidase studies were completec! with USC:of the Vectastain ABC kit. according to the procedures retommended by the manufacturer.” In brief. sections were incubated with a I :SO dilution of normal rabbi! s~*rum for

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30 minutes at room temperature, and excess serum was blotted from sections. Next, sections were incubated with either a 1: 100 dilution of rat anti-mouse IL-6 monoclonal antibody (1 mgiml) or a similar concentration of rat IgG (control) for 30 minutes at room temperature and washed twice. Sections were then incubated with a 1:200 dilution of biotinylated antirat IgG for 30 minutes at room temperature and washed twice. Next, sections were incubated with a peroxidase-conjugated avidin for 1 hour at room temperature and washed twice. The freshly prepared peroxidase substrate, 3.3 ’ diaminobenzidene tetrahydrochloride in 0.01% hydrogen peroxide, was incubated with specimens for 5 to 10 minutes. Sections were washed with tap water, dehydrated, and mounted under glass coverslips with use of Permount.

Cyclosporin

A administration.

CsA was dissolved in anhydrous alcohol to a final concentration of 60 mg/ml and stored at 4” C until use. Before administration, the stock solution of CsA was diluted 1: 100 in 0.9% normal saline solution. Individual mice were injected intradermally with anti-DNP IgE as described above. CsA (120 pg), in 200 ~10.9% normal saline solution containing 1% alcohol (see above) was given by tail vein injection 24 hours later. Mice receiving 200 pl of 0.9% normal saline solution with 1% alcohol served as controls. DNP,.,-HSA in PBS with 1% Evans blue was administered 3 hours later intravenously, mice were killed 2 hours after antigen injection, and skin biopsies were processed as above for Northern blot analysis. Treatment of mice with rmIL-6. In some experiments, mice received 100 units rmIL-6 in combination with antiDNP IgE in 20 p1 of PBS intradermally or anti-DNP IgE alone. Antigen with 1% Evans blue was administered 24 to 48 hours later, and areas of blueing (vasopermeability) were quantitated by outlining the blued area in pen, transferring the tracing onto translucent tape, which was then placed onto paper. The blueing was compared by weighing the cutouts of the blued area. In other experiments, mice received 500 U rmIL-6 in PBS or PBS alone by tail vein injection 30 minutes before intradermal sensitization with anti-DNP IgE. Antigen was again administered 24 to 48 hours later and the blued areas quantitated as above. In some experiments, mice similarly treated were killed 8 hours after antigen injection, the injected sites fixed in 4% paraformaldehyde, embedded in paraffin, and 2 to 5 pm sections were stained with Giemsa or hematoxylin and eosin to quantitate the cellular infiltrate. To assess the ability of rmIL-6 to elicit a blueing reaction or a cellular infiltrate, 200 U of rmIL-6 or PBS alone was injected intradermally. This was immediately followed by a tail vein injection of 1% Evans blue in PBS. Mice were killed 10 minutes later and the area of blueing assessed. In some instances, biopsies were obtained at the injection site and stained as above with Giemsa or hematoxylin and eosin to quantitate the cellular infiltrate. Statistical analysis. The results of blueing reactions or cell counts were analyzed for statistical significance

J ALLERGY

CLIN IMMUNOL NOVEMBER 1992

(p < 0.05) by Student’s t test for summary data. All results are expressed as the mean 2 SEM.

RESULTS Detection of IL-6 mRNA in mouse skin by Northern blot analysis during PCA To analyze the induction of mRNA for proinllammatory cytokines in mouse skin we used PMA, which is known to induce proinflammatory cytokine gene expression in vitro. PMA functions in part via protein kinase C and when applied topically to mouse skin promotes epidermal hyperplasia and tumor formation. 13,I4 It is known that several cells resident in the skin including keratinocytes,‘“, I6 mast cells,lA Langerhans cells, “, I8 and macrophages” transcribe IL- 1, IL-6, and TNF-o when treated with PMA in vitro. For these reasons, we made use of the topical application of PMA in DMSO to mouse skin as a positive control for cytokine production. Initial screening by Northern blot analysis consistently revealed only IL6 mRNA production (Fig. 1). IL-6 mRNA was detected 2 hours after application of PMA in DMSO and was still present 4 hours after topical PMA in DMSO. Application of DMSO alone did not result in detectable IL-6 mRNA in autoradiograms exposed for 40 hours. When autoradiograms were exposed for 96 hours, small amounts of IL-6 mRNA were detected in the DMSO control suggesting that although PMA was responsible for most IL-6 mRNA detected, application of DMSO to mouse skin is capable of inducing a small amount of IL-6 mRNA (data not shown). We next evaluated the effects of intradermal compound 48 I80 on proinflammatory cytokine production in mouse skin. Compound 48/80 is a polycationic polyamine that induces mast cell degranulation by a noncytolytic mechanism believed to occur through an ill-defined polyamine receptor found on mast cells.2o In addition to causing release of preformed mediators, it results in a mixed inflammatory cell infiltrate after intradermal injection in humans, similar to a latephase reaction.“’ 22 To date cytokine induction by compound 48/ 80 has not been investigated. Initial screening revealed only the consistent appearance of IL-6 mRNA. As shown in Fig. 1 (lanes 1 and 2), IL6 mRNA was detected 2 hours after intradermal injection of compound 48 180, and was no longer present 4 hours after injection. Intradermal injection of 20 p,l of PBS alone did not result in detectable IL-6 mRNA in autoradiograms exposed for 40 hours. When autoradiograms were exposed for 96 hours, small amounts of IL-6 mRNA were detected in the PBS control (data not shown). An in vivo model of PCA was next used to analyze

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IL-6 production

FltJri?(: PCX

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.’

FIG. 1. Northern blot analysis of IL-6 mRNA production in mouse skin after intradermal administration of compound 48180 at 2 hours (lane 1). and 4 hours (lane 2); topical PMA in DMSO at 2 hours (lane 3). and 4 hours (lane 4); intradermal anti-DNP IgE alone (lanes 5 and 6); and during PCA at 2 hours (lane 7), and 4 hours (lane 8) after antigen activation. Each lane contains RNA isolated from the skin of one mouse. Actin mRNA was assayed to ensure that relatively equal amounts of RNA were loaded into each lane. Experiment shown performed twice with identical results. Autoradiogram exposed for 40 hours.

proinflammatory cytokine production during a specific IgE-mediated reaction in BALB/c mouse skin. The PCA reaction that occurs in mouse skin is an IgEmediated mast cell-dependent event.6 Again, among the proinflammatory cytokines, only IL-6 mRNA was consistently identified at the sites of PCA. As shown in Fig. 1 (lanes 7 and 8), IL-6 mRNA was detected 2 hours after antigen activation and was no longer detected 4 hours after activation. No IL-6 mRNA was detected in skin that had been sensitized with antiDNP IgE only ( Fig. 1, lanes 5 and 6). To further analyze the kinetics of IL-6 mRNA production at PCA sites after antigen activation, skinsensitized mice were killed at 1, 2, 4, and 8 hours after antigen administration, and isolated RNA was subjected to Northern blot analysis. As shown in Fig. 2, A, IL-6 mRNA appeared as early as 1 hour after antigen activation (lane 3), but disappeared by 4 and 8 hours (lanes 5 and 6) after activation. No IL-6 mRNA was detected in untreated mouse skin (lane 1) or in mouse skin that had been injected with anti-DNP IgE only (lane 2). We next determined whether the appearance of IL6 mRNA was affected by treatment with CsA. Mice were injected intradermally with anti-DNP IgE followed by 6.0 mg/kg (120 kg) of CsA or diluent alone intravenously, 24 hours after anti-DNP IgE skin sensitization. A second control population of mice consisted of those that received neither diluent or CsA. Three hours after CsA treatment, DNP,,,,-HSA was given intravenously to each of the three groups. Animals were killed 2 hours after intravenous DNP30.40HSA administration, and IL-6 mRNA production was

examined by Northern blot analysis. As shown in Fig. 2, B (lanes 1 and 2), IL-6 mRNA was detected at 2 hours in PCA sites from animals that were not treated. IL-6 mRNA was also seen in an animal treated with diluent alone (Fig. 2, B, lane 3) as well as two separate animals treated with CsA (Fig. 2, B, lane 4 and 5). CsA treatment did not inhibit the increased vasopermeability seen as evidenced by extravasation of Evans blue at the site of the PCA reaction (data not shown). Therefore in this IgE-mediated, mast cell-dependent PCA reaction, parenteral GSA administration did not inhibit either the increased permeability associated with the immediate hypersensitivity reaction or the induction of IL-6 mRNA. Detection of IL-6 mf?NA during situ hybridization

PCA by in

We next sought to determine the location of cells producing IL-6 by examination of IL-6 mRNA production within tissue slices using in situ hybridization. On the basis of the kinetics of IL-6 mRNA expression by Northern blot analysis, we examined skin 2 hours after antigen injection. As shown in Fig. 3, panels A and B, when examining normal skin with a cDNA probe for IL-6, no cells within the skin section were identified that had greater than five grains overlying a single cell. However, 2 hours after ultravenous DNPloa-HSA, one to three cells per high-power field had greater than 10 grains over each c&l ( Fig. .J, panels C through F). These cells were primarily located within the dermis and possessed a11oval-roround nucleus without obvious granules. suggesting that they were either dendritic cells il,angerhans cells

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A

123456 IL-6

Actin

CLIN IMMUNOL NOVEMBER 1992

travenous DNP,,,-HSA. Increased staining was also seen in cells in the basal keratinocyte layer. The cells within the dermis again had an oval-to-round nucleus, suggestive of a dendritic cell, lymphocyte, or mast cell as the source of the IL-6 protein. No specific staining of either epidermal or dermal cells was detected with use of a similar dilution of rat IgG as the control antibody on a serial skin section (Fig. 4, B). In addition, when examining normal mouse skin, no specific staining was detected with use of the antimouse-IL-6 antibody (data not shown). Recombinant mlL-6 increases the vasopermeability seen during PCA

I3 12345 IL-6

P,-Microglobulin FIG. 2. A, Northern blot analysis of IL-6 mRNA at serial time points in mouse skin during PCA. Untreated skin (lane 1); anti-DNP IgE alone (lane 2); 1 hour after DNP 304,,-HSA (Ag) (lane 3); two hours after Ag (lane 4); four hours after Ag (lane 5); eight hours after Ag (lane 6). Each lane contains RNA isolated from the skin of one mouse. Experiment performed three times with identical results. Actin mRNA assayed to ensure that relatively equal amounts of RNA were loaded into each lane. B, Effect of systemic CsA on IL-6 mRNA production in mouse skin during PCA. Northern blot analysis of mRNA from skin 2 hours after intravenous DNP,,.,,-HSA in mice that did not receive intravenous diluent or CsA (lane 1 and 2); or received diluent alone (lane 3); or received CsA (lanes 4 and 5). p,-microglobulin mRNA was assayed to ensure that relatively equal amounts of RNA was loaded into each lane. Data shown are a composite of two experiments, with lanes 1, 3, and 5 from experiment one and lanes 2 and 4 from experiment two.

or macrophages), lymphocytes, or degranulated mast cells. lmmunohistochemical identification of IL-6 within the dermis during PCA reactions To determine if IL-6 protein is present during a PCA reaction and to investigate its source we next looked for immunoreactive IL-6 using an immunoperoxidase technique described above. As shown in Fig. 4, A, perinuclear and cytoplasmic staining was seen in cells throughout the dermis 4 hours after in-

To examine the possible biologic function of localized cutaneous production of IL-6 during a PCA reaction, we first injected rmIL-6 into mouse skin and then immediately followed this with intravenous Evans blue in PBS to see if rmIL-6 alone can cause increased vascular permeability. No blueing reaction occurred at the site of the rmIL-6 injection. When a biopsy was performed on the injection site 8 hours later and compared with skin sites injected with PBS alone, no significant difference was seen in the cellular infiltrate (rmIL-6: 36.7 ? 3.08 cells/HPF [X lOOO] vs PBS: 36.3 t 2.9 cells/HPF [X lOOO],p < 0.35). We next determined if local injection of rmIL-6 would affect the blueing reaction seen during the course of the PCA reaction. One hundred units of rmIL-6 were injected intradermally in combination with anti-DNP IgE. Twenty-four hours later, mice were administered DNP,,,,-HSA with 1% Evans blue, killed 5 minutes later, and the vasopermeability at the PCA site was quantified (Table I). Increased vasopermeability was evident in the five animals given rmIL-6 locally, and this was statistically significant when compared with the vasopermeability seen in the five animals not receiving rmIL-6. To determine whether systemic administration of rmIL-6 would affect the PCA reaction, we injected 500 U of rmIL-6 intravenously approximately 30 minutes before sensitization of the mice skin with anti-DNP IgE. Twentyfour hours later mice were administered intravenous DNP,,.,-HSA, killed 5 minutes later, and-the vasopermeability was quantitated. As shown in Table I, an increased vasopermeability reaching statistical significance was seen in each of three experiments when compared with animals that did not receive systemic intravenous rmIL-6. The administration of endotoxin, LPS, (S. minnesotu) intravenous or PBS alone, in place of rmIL-6 did not increase vasopermeability. (PCA alone: 32.3 & 8.4 mg, vs PCA and PBS: 29.4 k 7.8 mg, vs PCA & LPS: 26.6 ? 2.2 mg; p values cO.25).

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IL-6

FIG. 3. Detection of IL-6 ml?NA by in situ hybridization in mouse skin IL-6 cDNA probe, (A) normal skin (not sensitized), light field (x400) sensitized), dark field (x400); skin from a PCA reaction site (C) light field (x400), and same site, higher magnification, (E) light field (x (x 1000) 2 hours after intravenous DNP,,,,-HSA.

TABLE I. Increased Experiment I 3i 3 4

vasopermeability Route of IL-6 delivery

Intraderrnal Intravenous Intravenous

Intravenous

during

PCA after

Paper weight (mg) Ag + IgE 28.4 15.2 40.7 12.3

c +L k

2.5 0.9 6.8 0.1

(5) (5) (5) f,5)

local

or systemic

during PCA by use of ar! and (B) normal skin (not field ( x 400) and (D) dark IOOO), and (F) dark field

rmlL-6

Paper weight (mg) Ag + IgE + IL-6 64.1 52.9 77 21.4

i_ f -5 c

10.4 16.5 IS.8 O.Ol

production &rrnq KY% 821

(5) (5) (5) (5)

administration Percent increase

p value

125% 248% 8% 73%

.:O.Oi 3).05 :0.025 -:ri.os

--

Mice treated with either 100 units of rmIL-6 intradermal (experiment l), or 500 units of rmIL-6 mtravenous (experiment 2. 1. and 4). the blueing reaction quantitated, and compared with mice that had not been treated. Data are presented as the mean t SEM inj. uf single reaction sites from each of 5 mice per experiment. Statistical analysis performed hy use of Student’s I test for summary data

To determine whether rmIL-6 administration affected the late-phase cellular infiltrate, a second group of animals were killed 8 hours after intravenous administration of DNP,,.,-HSA, and the PCA areas were examined for a cellular infiltrate. No significant difference was observed in the cellular infiltrate in the rrnlL-6 treated animals (62.2 ? 5.9 cells/HPF [ x lOOO]) as compared with the animals that did not

receive rmlL-6 p < 0.3).

(58.6 2 4.0 cellsiHPF

I x ItIOO\.

DISCUSSION In vitro studies of BMCMCs and mast cell lines have shown that when these cells are activated through crosslinkage of Fc,RI or after PMA-mediated signals, mRNA for several proinflammatory cytokines can be

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CLIN IMMUNOL NOVEMBER 1992

FIG. 4. Detection of immunoreactive IL-6 protein in mouse skin during PCA by immunohistochemistry with use of a rat antimouse IL-6 antibody (x 630) (A) or rat IgG as a control ( x 630) (B) in skin processed 4 hours after intravenous DNP,O.,,-HSA.

detected. These proinflammatory cytokines, including IL-l, IL-6, TNF-a, MIP-la, MIP-lp, TCA-3, and JE, are known also to be produced by other cells including macrophages and lymphocytes.“, 19.23-25 Several of these cytokines are known to be chemotactic for neutrophils and macrophages.‘6-‘0 On the basis of this information it was our hypothesis that proinflammatory cytokines might be produced during the inflammatory response that follows mast cell activation in vivo. To explore this hypothesis, we obtained tissue from sites of PCA, a reaction known to be mast cell-dependent, as well as from sites of mast cell degranulation induced by compound 48 I80 and also from skin sites receiving topical application of PMA in DMSO as a positive control for cytokine production. The tissue samples were screened by Northern blot analysis for the presence of proinflammatory cytokine mRNA. Initial studies identified only IL-6 mRNA at skin sites exposed to PMA in DMSO and compound 48/80 and at sites of PCA (Fig. 1). TNF-a mRNA as reported previously was not seen in our studies possibly because of technical differences.’ The identification of IL-6 mRNA at sites of PCA agrees with the data showing that IL-6 can be identified in skin blister fluid at sites of allergic reactions in humans.’ IL-6 mRNA was detected by Northern blot analysis of cultured mast cell populations as early as 30 minutes and was no longer seen after 4 hours.3’ We thus performed a time course to determine the appearance of IL-6 mRNA during PCA. As shown in Fig. 2, A, IL-6 mRNA is seen at 1 and 2 hours. IL-6 is known to be produced in vitro by many different cells resident in the skin, including keratin-

ocytes, endothelial cells, and fibroblasts.23 In fact the ability to easily demonstrate IL-6 mRNA may be due to production of IL-6 by other cells stimulated during the allergic reaction. This would be one explanation for the identification by Northern blot analysis of IL6 mRNA in the absence of mRNA for the other proinflammatory cytokines, which may be more restricted in their cells of origin. The time of appearance of IL-6 is against but does not exclude the possibility that cells entering the skin may contribute to its production. In situ hybridization revealed that one to three cells per high-power field were producing IL-6 mRNA (Fig. 3). Similarly, immunohistochemistry (Fig. 4) revealed cells in the basal keratinocyte layer and dermal cells producing immunoreactive IL-6 protein. In some cases dermal cells identified by in situ hybridization appeared to have a round-to-oval nucleus, suggesting dendritic cells (Langerhans cells or macrophages), degranulated mast cells, or lymphocytes. To date, studies to definitively identify these cells have not been successful. The inability to identify cells in the basal keratinocyte layer by in situ hybridization that produce IL-6 mRNA is probably due to the difference in sensitivity of this technique when compared with immunohistochemical studies identifying IL-6 protein. The ability to identify IL-6 mRNA by Northern blot analysis at the sites of PCA and the inability to consistently identify mRNA for the other proinflammatory cytokines does not exclude their presence at sites of PCA. For instance, the ability to detect mRNA by Northern blot analysis is less sensitive than the ability to identify mRNA through reverse transcription in

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combination with the polymerase chain reaction. The variability seen in signal intensity for IL-6 mRNA in Figs. 1, 2, A, and B may reflect technical variability from day to day or variability in the degree of mast cell activation between mice. However, the ability to easily and consistently identify IL-6 mRNA at sites of PCA in each of three experiments performed, as well as immunoreactive IL-6 proteins, does unequivocally demonstrate that IL-6 must be considered as one of the mediators identified in the inflammation that follows the allergic response in the skin. IL-6 is a multifunctional cytokine often classified as proinflammatory because of properties it has in common with the proinflammatory cytokines, IL-l and TNF-a. However. although IL-6 is able to induce fever in rabbits and cause bone resorption,32. 33many of its properties are appropriately classified as antiinflammatory. Biologic effects of IL-6 include activation of T and B lymphocytes,34. 35 stimulation of immunoglobulin production by B cells,26 and the induction of acute-phase protein synthesis by not only hepatocytes but also monocytes, fibroblasts, and endothelial cells. i7, ‘* IL-6 lacks many of the proinflammatory functions of IL- 1 and TNF-c~, and in macrophages IL6 has been shown to diminish lipopolysaccharide-induced TNF-c~ and IL- 1 production.” To investigate the possible biologic consequences of local IL-6 production during an IgE-mediated mast cell-dependent reaction in mouse skin, we next injected rmIL-6 alone into mouse skin and found that it did not increase vasopermeability or appear to alter the composition or time course of appearance of the cellular infiltrate. Injection of rmIL-6 during a PCA reaction. either intradermally at the PCA site or intravenously during skin sensitization, resulted in an increased blueing reaction. It is interesting to note that when basophils are cultured with IL-6, increased amounts of histamine are released in response to Fc,RI signals.J” But even in the presence of an increased blueing response (with rmIL-6), the cellular infiltrate remained unchanged 8 hours after antigen administration. Although the role of IL-6 in the allergic response remains unclear, its ability to induce acute-phase protein synthesis both locally and within the liver may be important to this process. In a study of insect stinginduced anaphylaxis, Smith et a1.4’ noted a rise of fibrinogen (an acute phase protein) above baseline in two patients after an insect sting. Mice undergoing experimental contact sensitivity reactions have also been found to develop elevated levels of two acutephase proteins, haptoglobin and semm amyloid A, and the degree of elevation was correlated with the amount of increased skin swelling measured.4’ It is thus possible that during these inflammatory reactions, ele-

during

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vated IL-6 levels induced these acute-phase responses. Induction of local IL-6 synthesis and subsequent rclease into the blood during allergic reactions. thus in the aggregate, may. be one mechanism by which an allergic reaction if sufficiently widespread ~I:OUId !ead to systemic acute-phase response. CsA is an antiinflammatory compound and has been suggested as one drug that may modulate ailergic disease. CsA inhibits histamine release from rat and human mast cells and human basophils in vitro.” ‘-’ However, CsA does not inhibit IL-6 mRN;2 production after Fc,RI activation in murine mast ceil lines.’ To determine whether CsA would alter lL-6 mRNA production during the PCA reaction. WCnext administered CsA at therapeutic doses to mice followed by induction of PCA.45 As shown in Fig. 1. IS, GA administration did not alter IL-6 mRNA production at sites of PCA. In addition, CsA did not effect the vascular permeability at sites of PCA as a!,sessedby the blueing reaction. Thus in the PCA reaction CsA had no effect on either the immediate reaction as a$sessed by blueing or the production of’ IL-h mRN.4. Thus we have demonstrated that LL-6 rnKNA is produced in skin after IgE-mediated mast cell dcgranulation, and this induction is not inhibited by C.;A administration. IL-6 protein is produced within 4 hours and is associated with cells throughout the dermis and cells in the basal keratinocyte layer Although the biologic relevance of IL-6 production :it sites of PCA remains unclear, its administration does et’fect permeability, and IL-6 does have the unique property of promoting systemic acute-phase reactlcjn if produced in sufficient amounts. IL-6 thus jiyins histamine, prostaglandins, leukotrienes, P-&F. and TNF-cx as one of the many mediators generated during an allergic reaction. The authors thank Mrs. Belinda itorial assistance.

Rich&son

i‘or her cd-

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