Arch Dermatol Res (1990) 282: 318 - 324 Archives of

�9 Springer-Verlag J 990

Effects of interferon-v treatment on the cutaneous DT H reaction in rats

C. Skoglund and A. Scheynius

Department of Clinical Immunology, Karolinska Institute, S-104 01 Stockholm, Sweden

Received November 18, 1989

Summary. The capacity of interferon-v (IFN-J to induce class I I histocompatibility antigens on different cell types including keratinocytes, is well known, but the impact of IFN-v on the immune response is still unclear. Lewis rats sensitized with dinitrofluorobenzene (DNFB) were injected with recombinant rat IFN-? (10 s U) or phosphate-buffered saline (PBS) once daily on 3 successive days at the bases of the ears either before or after they were challenged on the ears. As expected, the PBS-treated animals showed about a 30% increase in ear thickness and there was an induced expression of class I I antigens on the keratinocytes as judged by immunohistochemistry 72 h after challenge. Exogenously added IFN-v prior to DNFB challenge result- ed in a significantly reduced ear swelling at 24 (p < 0.01) and 48 h (p < 0.05) after challenge. In this case the keratin- ocytes expressed class I I antigens already at the time of challenge. When IFN- 7 injections were given during the contact allergic reaction there was no significant reduction of ear swelling until 72 h (p < 0.01). At that time point there was a more pronounced expression of class I I antigens on the keratinocytes compared with PBS-injected animals, due to the IFN-v treatment. These in vivo data support our previous observations that IFN-v may play a self-limiting role in certain immune responses.

Key words: Class II antigens - Contact allergic reac- tion - In te r fe ron-gamma - Ra t skin

Cutaneous immunological reactions comprise a complex system of regulatory mechanisms with changes in cell populat ions and intriguing interactions between regula- tory substances. Induced expression of class II histo- compatibil i ty antigens on keratinocytes is a striking fea- ture in several conditions in the skin, characterized by T- cell infiltration [12, 20, 29]. It has been suggested that this phenomenon is mediated mainly by interferon- 7 (IFN-7)

Offprint requests to: Dr. C. Skoglund

[2, 21], a cytokine produced by activated T cells [15]. Apar t f rom this class II inducing ability, IFN-7 is known to function as a macrophage activating factor [23] and as a possible regulator of inf lammation [3].

Previously we have shown [25] that in vivo treatment of rats with a mouse monoclonal ant ibody (MoAb) di- rected against rat IFN-7 inhibited the normally induced expression of class II antigens on keratinocytes during a delayed type of hypersensitivity (DTH) reaction and led to an enhanced local reaction. Based on these obser- vations, we postulated that under certain conditions, IFN-7 may play a self-limiting role in the immune re- sponse.

In the present study recombinant rat IFN-? was inject- ed locally before or during the elicitation of a contact allergic reaction in rats to investigate further the in vivo effects of this cytokine on the development of a cutaneous D T H reaction.

Materials and methods

Animals

Three-to five-month-old Lewis rats of either sex obtained from Anticimex (Stockholm, Sweden) were fed with pellets and allowed free access to water.

Sensitization and elicitation of contact sensitivity

In a 4:1 mixture of acetone and olive oil (F. Merck, Darmstadt, FRG and Sigma Chemical, St. Louis, Mo., USA) 20 gl of 0.5% 2,4- dinitro-1-fluorobenzene (DNFB) (Sigma) was painted on the shaved abdominal wall skin on days 0 and 1. On day 5 the animals were challenged by applying 20 gl of 0.2% DNFB, in the same vehicle, on the dorsal side of the left ear while the right ear was painted with the vehicle only.

IFN-y treatment

Rat recombinant IFN-7 was generously provided by Dr. P. H. van der Meide, TNO, Primate Centre, Rijswijk, The Netherlands [5],

C. Skoglund and A. Scheynius: IFN-y effects on DTH 319

Table 1. Effects of intradermal injections of IFN- 7 (105 U) in a volume of 0.1 ml once daily on two or three consecutive days at the bases of the ears

No. of No. of Central longitudinal sections of the ears injections ears

Class II antigen expression on keratinocytes ~ Dermal cell infiltrate b

Proximal 1/4 Middle 2/4 Distal 1/4 Proximal 1/4 Distal 3/4

2 4 + + + + - 2 0 - 1 3 4 + + + + + + 2 0 - 1

a Expression measured by immunoperoxidase staining with MoAb OX6 on frozen sections of the ears taken 24 h after the last injection and graded as: - undetectable, + single groups of keratinocytes, + + continuous and covering the basal layer, and + + + continuous and extending throughout the whole epidermis b Estimated on H & E stained sections and graded as: 0, none; 1, small; 2, moderate; 3, large

Table 2. Effects of IFN- 7 on the ear swelling

No. of rats Injection at sensitized the base of the to DNFB at ear at days day 0 and 1

Percentage ear swelling (mean • SEM) compared with day 5 before challenge

Hours after challenge

24 48 72

Experiment 1 7

Experiment 2 14

10

IFN- 7 25.1 _+ 2.9** 27.2 +_ 4.1 * 24.1 + 2.6 (105 U) 2 , 3 , 4

PBS 37.9 _+ 2.5 38.4 +_ 1.0 30.7 • 2.0 2 , 3 , 4

IFN-7 33.1 + 3.7 24.2 +2.2 14.3 _ 1.9"* (105 U) 5 ,6 ,7

PBS 36.8 _+ 2.1 30.4 _+ 2.6 24.0 +_ 2.7 5 ,6 ,7

* Significantly different from PBS control, p < 0.05 (Student's t-test) ** Significantly different from PBS control, p < 0.01 (Student's t-test)

which was stored freeze-dried at + 4 ~ C and dissolved in phosphate- buffered saline (PBS) at pH 7.4 before using. Previously we had found that intradermal injections of 104 U IFN-7 into the dorsal surface of rat ears once daily on 3 successive days induce a strong expression of class II antigens on keratinocytes [21]. Since the inj ec- tions also result in local dermal cell infiltrates [21], which may impair the evaluation of ear swelling caused by the DTH reaction, we first tested different concentrations of IFN-~/and numbers of injections given intradermally at the base of each ear of unsensitized rats. As can be seen in Table 1, three daily injections of 105 U IFN- 7 in a volume of 0.1 ml resulted in an expression of class II antigens on keratinocytes throughout the whole ear on both the dorsal and ventral sides as judged by immunoperoxidase staining (see below). At the same time the dermal cell infiltrates, of moderate size, were located mainly to the proximal fourth of the ear (Table 1). This regimen was therefore chosen for the following experiments.

To investigate the effects of IFN-7 given locally before a DTH reaction, the DNFB challenge in sensitized rats was preceded by one daily injection of 0.1 ml IFN-7 (105 U) at 72, 48, and 24 h before challenge (Table 2, experiment 1). A control group of sensitized animals was treated in the same way except for injections with PBS alone (0.1 ml). In experiment 2 IFN-~ was administered during a

DTH reaction; DNFB-sensitized animals received one injection of IFN-7, concentration and volume as in experiment 1, immediately before DNFB challenge and also 24 and 48 h after (Table 2, experiment 2). Injections with 0.1 ml PBS were given to the control group according to the same protocol. For convenience this exper- iment was performed identically on two different occasions (8 + 6 and 6 + 4 animals respectively).

In both experiment 1 and 2 some additional DNFB-sensitized animals were only challenged as a control of the sensitizing regimen. No differences in ear swelling or class II antigen expression were seen as compared with the PBS-treated animals in any experiment, which was why the latter were used as the proper control. The groups were age- and sex-matched and the experiments were performed under ether anesthesia.

Evaluation o f ear thickness

The ear thickness was measured under ether anesthesia before chal- lenge at day 5 and also after 24, 48, and 72 h, using a digital micrometer (Mitutoyo, Tokyo, Japan). Each ear was measured at

320 C. Skoglund and A. Scheynius: IFN- 7 effects on DTH

Fig. 1 a, h. Immunoperoxidase staining of OX6 reactive cells on frozen sections of rat ear skin from DNFB sensitized animals, 72 h after painting with the vehicle alone. Note absence of class II antigen expression on keratinocytes after daily injections of 0.1 ml (a) PBS for 3 days and presence after (b) l0 s U IFN- 7 for 3 days. Sections are counterstained with Mayer's hematoxylin

Immunohistochemical staining

Acetone-fixed, 6-pm thick, longitudinal cryostat sections of the ears were processed for peroxidase-anti-peroxidase (PAP) staining ac- cording to the method of Sternberger [28]. The sections were cut through the center of each ear extending from the top to the base. Mouse monoclonal antibodies (MoAbs), clones OX6 and OX17 were obtained from Sera-Lab (Cambridge, UK). OX6 antibodies (IgG1, diluted 1/2,000) have been shown to react with constant region determinant of rat class II antigens corresponding to I-A coded molecules in mice [14], whereas OXl7 antibodies (IgG1, di- luted 1/1,000) recognize a monomorphic determinant on the e-chain of rat class II antigens, homologous with the mouse I-E product [6]. Besides OX6 and OX17 expression, the following mouse MoAbs were used: OX8 (IgG, diluted 1/320) (Sera-Lab), reactive with the rat counterpart to the CD 8 antigen on "suppressor/cytotoxic" T cells [4], W3/25 (IgG, diluted 1/50), reactive with the rat counterpart to CD 4 antigen on "helper-inducer" T cells and on some macrophages [1], produced and purified as earlier described [13], and OX42 (IgG2a, diluted 1/320) (Serotec, Oxford, UK) directed against most rat macrophages, dendritic cells, granulocytes and probably the rat homologue of the human iC3b receptor [19]. Goat anti-mouse IgG was adsorbed on Sepharose-coupled rat IgG and used as a secondary antibody (diluted 1/10) except for stainings with OX42 where a commercially available goat-anti-mouse IgG (ATAB, Scarborough, Me., USA) was used (diluted 1/40). Preformed complexes of horseradish peroxidase and monoclonal anti- horseradish peroxidase antibody (diluted 1/500) were obtained from Dakopatts (Copenhagen, Denmark). The peroxidase reaction was developed with 3-amino-9-ethyl carbazole [10] and the sections counterstained with Mayer's hematoxylin. The dilutions of the anti- bodies were determined using sections from normal lymph nodes and skin. Controls without the primary antibodies or with irrelevant antibodies gave no staining. The expression of class II antigens on the keratinocytes was graded from - to + + + , where - denotes undetectable, + single groups of keratinocytes, + + continuous and covering the basal layer, and + § + continuous and extending throughout the whole epidermis. Keratinocytes were considered positively stained when the full circumferences of the cells were labelled. The longitudinal sections were assessed in three parts; the proximal fourth, the middle part, and the distal fourth of the ear.

For estimation of dermal cell infiltrates each ear was also processed for hematoxylin and eosin staining. The longitudinal bi- opsy specimens were assessed as a proximal part (the proximal fourth) and a distal part (the remaining three-fourths), and the presence of cell infiltrates were classified as: 0, none; 1, small; 2, moderate; 3, large.

three separate loci around the lobe. The results are expressed as percentage ear swelling in each group (mean _+ SEM) as compared with day 5 (before challenge). To correct for injection and vehicle effects the values were calculated according to the following equa-

tion: ( a - b) - ( c - d) x 100, where a and b are the mean thicknesses b + d

2 of the left ear after and before DNFB challenge respectively, and c and d the corresponding figures for the right ear (vehicle pain- ted).

Specimens

After the final ear measurement at 72 h the rats were killed with ether. The ears were cut off at the bases, immersed in Histocon (HistoLab, Gothenburg, Sweden) at + 4~ (for maximally 2 h), snap-frozen in chilled isopentane, and stored at - 7 0 ~ C.

Statistical analysis

The statistical significance of differences in the mean of relative ear swelling for each experimental group was calculated with Student's t-test.

Results

Ear swelling

I F N - 7 t r e a t m e n t p r i o r to D N F B cha l l enge resu l ted in a s ign i f i can t ly r e d u c e d ear swel l ing a t 24 a n d 48 h c o m - p a r e d w i th the c o n t r o l g r o u p (Tab le 2, e x p e r i m e n t 1). W h e n I F N - 7 i n j ec t i ons were g iven d u r i n g the D T H reac- t i on there was n o s ign i f i can t r e d u c t i o n o f ear swel l ing u n t i l 72 h af ter cha l l enge (Table 2, e x p e r i m e n t 2).

C. Skoglund and A. Scheynius: IFN-7 effects on DTH 321

Fig. 2 a - e. Immunoperoxidase staining of OX6 reactive cells on frozen sections of rat ears from DNFB sensitized animals, 72 h after topical DNFB challenge. The painted, dorsal sides of the ears are oriented upwards. Note differences in ventral epidermis after daily injections of 0.1 ml (a) PBS for 3 days before challenge, (b) 105 U IFN-7 for 3 days before challenge, and (e) 105 U IFN-v for 3 days after challenge. Sections are counterstained with Mayer's hematoxylin

Expression of class H antigens in the epidermis

Epidermal expression of class II antigens in PBS-injected ears painted with vehicle alone was only detected on a few dendritic cells, presumably Langerhans' cells (Fig. l a; Table 3). Ears that received PBS injections prior to (experiment I) or during (experiment 2) DNFB challenge revealed class II expression dorsally throughout the whole epidermis in the middle part of the biopsy (Fig. 2a), with single groups of positive keratinocytes in the most distal and proximal parts (Table 3).

Animals treated with IFN-7 before DNFB challenge, expressed class II antigens on keratinocytes in challenged ears in a similar pattern as that in PBS-treated animals (Fig. 2b; Table 3, experiment 1). In IFN-,/ treated, un- challenged ears patchy staining on keratinocytes was lim- ited to the proximal fourth of the ear (Table 3, ex- periment I).

IFN- 7 applied exogenously during the DTH reaction (Table 3, experiment 2) caused a more widespread ex- pression of class II antigens compared with PBS-injected animals; positively stained keratinocytes were seen along the whole epidermis on both the dorsal and ventral sides of the ear (Fig. 2 c). The expression on unchallenged ears after IFN-7 treatment was similar to that seen when IFN- 7 injections were given to unsensitized rats once daily for 3 consecutive days (Fig. I b; Tables 1, 3).

The estimates above of class II transplantation anti- gen expression were made after staining with MoAb OX6. Induced expression of OX17 reactivity on keratinocytes was also observed but to a lesser extent.

Dermal cell infiltrates

In DNFB-challenged ears there were moderate to large dermal cell infiltrates located in the distal 3/4 of the ears

independently of the substance injected (Table 3 and Fig. 2). As can be seen in Table 3 there was a difference in the proximal fourth of the ears 72 h after challenge: the animals which had received IFN-7 injections had larger cell infiltrates compared with those given PBS. Furthermore, the cell infiltrates were somewhat larger in experiment 2 than experiment 1.

Phenotypic characterization of the dermal cell infil- trates revealed that the majority of cells, independent of treatment, expressed class II antigens, assessed[ both by MoAbs OX6 (Fig. 2) and OX17. W3/25 positive cells were always more numerous than OX8 reactive cells and the number of cells stained for OX42 were fewer than OX8. This pattern was independent of IFN-7 or PBS treatment or whether the injections were given before or during challenge. Cells reacting with OX42 consisted mainly of two types: dendritic or granulocyte-like, where the dendritic types outnumbered the granulocyte-like types in DNFB-challenged ears. In unchallenged ears there were no obvious phenotypic differences on dermal cells between IFN-~' or PBS injected animals.

D i s c u s s i o n

The aim of this study was to evaluate in vivo effects of IFN- 7 on a contact allergic reaction. We found that exogenously added IFN-~ prior to DNFB challenge re- sulted in a statistically significant reduced ear swelling at 24 and 48 h after challenge. In this case the keratinocytes expressed class II antigens already at the time of chal- lenge. Three days later the expression was similar to that found in the PBS-injected and DNFB-challenged rats, which is in accordance with a previous study [21] showing that the induced expression of class II antigens on kera- tinocytes disappears within 6 days after the last injection. When IFN-~ injections were given during the contact allergic reaction there was no significant reduction of ear

322 C. Skoglund and A. Scheynius: IFN- 7 effects on DTH

Table 3. Effects of IFN- 7 on the expression of class II antigens on keratiocytes and on dermal cell infiltrates 72 h after challenge

No. of rats Injection Substance sensitized at the applied to DNFB base of on the at day 0 the ear ears at and I at days day 5

Central longitudinal sections of the ears

Class II antigen expression a Dermal cell infiltrate b

Proximal I/4 Middle 2/4 Distal 1/4 Proximal 1/4 Distal 3/4

Experiment 1

7 IFN-y (1o 5 u) 2,3,4

6 PBS 2,3,4

DNFB (left) + / + + + + + + 1 - 2 2

a/o ~ (right) + - - 1 - 2 0

DNFB (left) + + + + - / + 0 - 1 2

a/o (right)

Experiment 2 DNFB (left)

14 IFN-7 (10 5 u) 5,6,7

10 PBS 5,6,7

+ + + + + + + + +

0 -1 0

2 2 - 3

a/o (right) + + + + + + 2 0 -1 DNFB (left) + + + + - / + 1 2 - 3

a/o (right) - - - 1 0

a Expression was measured by immunoperoxidase staining with OX6 and graded as: - undetectable, + single groups of keratinocytes, + + continuous and covering the basal layer, and + + + continuous and extending throughout the whole epidermis b Estimated on H&E stained sections and graded as: 0, none; 1, small; 2, moderate; 3, large c Acetone/olive oil

swelling until 72 h. At that time point there was a more pronounced increase of class I I antigens on the keratin- ocytes compared with PBS-injected animals, due to the IFN- 7 treatment. The outcome of exogenously added IFN- 7 on the contact allergic reaction is thus dependent on when it is administered.

The present in vivo data support our previous obser- vations that epidermis containing class II expressing keratinocytes may contribute to the down-regulation of an antigen-specific T cell response, and that IFN-7 may play a self-limiting role in certain immune responses. Thus, epidermal cell suspensions f rom rats treated with IFN-7 in vivo [26] or f rom tuberculin reactive areas [22], thereby containing class II expressing keratinocytes, can induce a lower antigen specific T-cell response in vitro compared with normal epidermal cells. These findings are supported by those of Gaspar i et al. who in the murine system have demonstrated that class I I positive keratinocytes are able to induce antigen-specific unres- ponsiveness in hapten-specific Th l clones [7]. Further- more, we have shown that systemic t reatment of rats with MoAbs directed against I F N - 7, if given at the same time as elicitation, inhibited the normally induced expression of class II antigens on keratinocytes during a D T H reac- tion and led to an enhanced local swelling [25]. We have proposed that this down-regulation need not depend on the induced keratinocyte expression of class II molecules per se but on an increased product ion of prostaglandin E2 (PGE2) [26], a known feedback inhibitor o f immune functions [8]. Since IFN-7 enhances the secretion of PGE2

by keratinocytes [17], this might contribute to the ob- served reduction of the ear swelling. Furthermore, IFN- 7 regarded as a macrophage activating factor (MAF) [23], may have stimulated the development of suppressive macrophages leading to a suppressed T-cell response [11]. The reduced ear swelling noted early during the D T H reaction in experiment 1 may thus be explained by the conditions already created by exogeneously added IFN- 7 before challenge.

One obvious effect of local IFN-7 injections besides inducing expression of class II antigens is that they result in a cellular infiltrate [21]. In this study we chose to give the injections at the bases of the ears so as to limit the distribution of cell infiltrates to the proximal parts of the ears. As seen in Table 1, class II expressing keratinocytes were still seen all along the longitudinal biopsy specimen after three injections although decreasing towards the periphery. This may indicate that the concentration of exogenous IFN- 7 was high enough to induce class II ex- pression on keratinocytes in the whole ear but below the threshold for attracting mononuclear cells in the distal parts of the ears.

When looking for phenotypic differences in the com- position of the dermal cell populations between IFN- 7 or PBS treated rats, 3 days after D N F B challenge, we did not detect any using MoAbs OX6, OX17, W3/25, OX8, or OX42. Nor was there any difference in the ears painted with acetone/olive oil alone. Thus, IFN-7 treatment does not seem to give rise to a phenotypically different cell response f rom that of a D T H reaction.

c. Skoglund and A. Scheynius: IFN-7 effects on DTH 323

Recent ly Sh iohara et al. [24] reported tha t pretreat- ment o f mice with mouse r ecombinan t IFN-7 given intra- dermal ly in the foo tpad before injection o f autoreact ive T cells at the locat ion enhanced the l ichenoid tissue react ion and increased the foo tpad swelling as c o m p a r e d with pre t rea tment with PBS. This m a y seem con t rad ic to ry to our f indings bu t their model resembles more an a u t o i m m u n e react ion where I F N - 7 t rea tment m a y have an enhancing effect. Pani tch et al. [18] have found that in patients with multiple sclerosis, in t ravenous infusion with IFN-7 increased the exacerbat ion rate o f the disease. W h e n IFN-~ was injected into the foo tpads o f mice dur- ing the deve lopment o f collagen II induced arthritis, the f requency and severity were higher and also resulted in an earlier onset [16]. In N Z B mice, with inbred lupus-like nephritis, the disease is worsened after systemic IFN-7 t rea tment but after regular injections with M o A b s to IFN-7 the survival rate after 11 mon ths was raised to 95% as c o m p a r e d with < 20% in PBS-treated animals [9]. Accord ing to Steiniger et al. [27] the maximal tissue concent ra t ion o f IFN-7 that can be achieved by systemic appl icat ion administered as con t inuous infusion o f rats is limited due to el iminat ion or inactivation. One or o ther o f these mechanisms m a y be dis turbed in a diseased state like au to immuni ty , which might explain the difference in response to I F N - y between the a u t o i m m u n e condi t ions and the D N F B - i n d u c e d react ion in normal rats.

The present data, taken together with our previous observat ions [22, 25, 26], s t rongly suppor t our hypothesis tha t I F N - 7 exerts effects which indicate a self-limiting role for this cy tokine in the cu taneous D T H reaction. The response to I F N - 7 seems to be differently regulated depending on whether an a u t o i m m u n e condi t ion or a D T H react ion in an undiseased individual is treated. Be- fore one can apply in vivo t rea tment with either IFN-7 or its M o A b s , more knowledge is needed abou t the effects o f IFN-y , as the ou t c om e seems to depend not only on the dose, the time at which it is given, or mode o f adminis t ra t ion, bu t also on the pathological state.

Acknowledgements. Excellent technical assistance was given by Ms. H. Nilsson, Ms. C. Johansson, and Ms. K. Johansson. This work was supported by grants from the Swedish Work Environment Fund, the Swedish Medical Research Council, the Foundation of King Gustav V, and the Edvard Welander Foundation.

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