LCB 2183 inhibits the inflammation associated with oxazolone-induced contact sensitivity

Int. J. lmmunopharmac., Vol. 16, No. 8, pp. 675-683, 1994 Elsevier Science Ltd International Society for lmmunopharmacology Printed in Great Britain 0192-0561/94 $7.00 + .00

Pergamon 0192

-

0561(94)E0028-L

LCB 2183 INHIBITS THE I N F L A M M A T I O N ASSOCIATED WITH O X A Z O L O N E - I N D U C E D CONTACT SENSITIVITY N. MURRAY,*t N. ZOERKLER,* T. BROWN* and Y. BONHOMME§ *Lipha SA, Rue des Bosquets 6,1800 Vevey, Switzerland; *Lipha Pharmaceuticals, High Street, West Drayton, Middlesex UB7 7QG, U.K.; and ~Lipha R & D, 34 rue St Romain, 69379 Lyon Cedex 08, France

(Received for publication 24 February 1994)

- - LCB 2183, an anti-allergic and potential anti-asthma compound, has been investigated for its ability to inhibit contact sensitivity in the mouse. The delayed response to epicutaneous hapten challenge in this model is a classical T-cell-mediated inflammatory reaction which is dependent on an early initiation phase. Both the early and late components of oxazolone-induced contact sensitivity were inhibited by oral administration of LCB 2183 in a dose-dependent manner. The drug appears to act on the efferent limb of the response since administration before hapten challenge was effective, while administration before the initial sensitization was not. LCB 2183 acts early in the cascade of events leading to inflammation, since the initiation phase of the response was inhibited; nonetheless, an effect of the drug on the late acting inflammatory cells cannot be ruled out. In comparison with oral prednisolone, which was also able to inhibit both the early and late components of the response, LCB 2183 was less active. Sodium cromoglycate and nedocromil sodium, which are poorly absorbed from the gastrointestinal tract, were tested by intraperitoneal administration. Neither of these agents significantly altered the delayed response and only nedocromil sodium had a limited inhibitory effect on the early initiation phase. Thus, in this model, LCB 2183 demonstrated more anti-inflammatory potential and resembled prednisolone more closely than either nedocromil sodium or sodium cromoglycate. The possible relevance of these effects in relation to the inflammation which characterizes human asthma is considered.

Abstract

Contact sensitivity is a variant of delayed-type hypersensitivity (DTH) which is induced by epicutaneous sensitization and challenge with reactive haptens such as oxazoione (Gocinski & Tigellar, 1990). Contact sensitivity has been extensively studied in the mouse and there is a great deal of information concerning the mechanisms involved in induction (Sullivan, Bergstresser, Tigelaar & Streilein, 1986; Welsh & Kripke, 1990; Cruz, Tigelaar & Bergstresser, 1990; Matsushima & Stohlman, 1991; Gaspari & Katz, 1991), expression (Gocinski & Tigelaar, 1990; Askenase, Bursztajn, Gershon & Gershon, 1980; Van Loveren, Meade & Askenase, 1983; Van Loveren et al., 1984; Van Loveren & Askenase, 1984; Ptak, Herzog & Askenase, 1991) and regulation (Ptak, Gershon, Rosenstein, Murray & Cone, 1983; Flood, Ptak & Green, 1986; Girolomoni & Tigelaar, 1990; Herzog, Ptak & Askenase, 1990; Gautam, Chikkala &

Hamilton, 1992) of the response. The expression of the delayed inflammatory response to hapten challenge requires the recruitment of antigen-specific T-cells which subsequently recruit non-specific inflammatory cells (Van Loveren, Meade & Askenase, 1983; Van Loveren et al., 1984). T-cell recruitment is brought about by an antigen-specific early initiation phase (Van Loveren & Askenase, 1984). This early initiation phase can, under certain circumstances, be mediated by low concentrations of IgE (Ptak, Geba & Askenase, 1991) or, more typically, by non-IgE initiation factors (Ptak, Askenase, Rosenstein & Gershon, 1982; Askenase, Rosenstein & Ptak, 1983; Van Loveren et al., 1986). These factors are produced by primitive, thymusindependent cells (Herzog, Meade, Pettinicchi, Ptak & Askenase, 1989; Herzog, Ferreri, Ptak & Askenase, 1989; Herzog, Millet, Ferreri, Ramabhadran, Schreurs & Askenase, 1990) and have been

tAuthor to whom correspondence should be addressed. Present address: LIPHA SA, 115 Avenue Lacassagne, F-69003 Lyon, France. 675

676

N. MURRAYet al.

shown to be involved in contact sensitivity (Herzog, Millet, Ferreri, Ramabhadran, Schreurs & Askenase, 1990), D T H (Herzog, Meade, Pettinicchi, Ptak & Askenase, 1989), graft rejection (Van Loveren, De Weger, Garssen, Los & Askenase, 1989) and autoimmune models (Askenase, 1992). Of particular interest, from the point of view of the present work, is the fact that in a DTH-like response in the mouse lung (Garssen, Nijkamp, Wagenaar, Zwart, Askenase & Van Loveren, 1989) this early initiation phase has been shown not only to induce the recruitment of antigen-specific T-cells but also to provoke an increase in airway resistance and airway hyper-reactivity (Garssen, Nijkamp, Van der Vliet & Van Loveren, 1991). Human asthma is a disease characterized by increased airway resistance and airway hyperreactivity as well as inflammation (Barnes, 1989). It is now well established that activated T-cells play an important role in the inflammation associated with asthma (Corrigan, Hartnell & Kay, 1988; Walker, Virchow, Bruijnzeel & Blaser, 1991; Brown, Crompton & Greening, 1991). How these T-cells are recruited and exactly how they influence the recruitment and activation of eosinophils and other cells remains to be established. Clearly, in allergic asthma, IgE-mediated events could lead directly to recruitment of both T-cells and eosinophils. Alternatively, IgE-mediated events could lead to T-cell recruitment and, subsequently, to eosinophil recruitment via T-cell activation. In other circumstances, for example non-allergic asthma, T-cell recruitment is not IgE-mediated and the possibility that non-IgE initiation factors are involved should be considered (Askenase, 1992). Increasingly the treatment of asthma is being directed at the underlying inflammation and corticosteroids are considered to be the most effective therapy currently available (Barnes, 1989). Recently, it has been shown that drugs which can inhibit the activation of recruited T-cells are effective in the management of asthma (Alexander, Barnes & Kay, 1992). Drugs which act earlier in the inflammatory cascade may be even more valuable. Perhaps some of the clinical effects of anti-allergic drugs, such as sodium cromoglycate and nedocromil sodium, are related to inhibition of IgE-mediated T-cell recruitment. In the present study we have investigated whether LCB 2183, an anti-allergic and potential anti-asthma drug currently in phase II of clinical development, could inhibit oxazolone hypersensitivity in the mouse ear. Two independent protocols were used to investigate drug effects on the early initiation phase and

the classical delayed hypersensitivity response. For comparison we have also examined the effects of prednisolone, nedocromil sodium and sodium cromoglycate. These drugs are all widely used in the management of asthma.

EXPERIMENTAL PROCEDURES

Materials The test product, LCB 2183 batch number HDS 1393 C, was supplied by Lipha (France). The standard anti-allergic compounds, nedocromil sodium and sodium cromoglycate were obtained as commercially available preparations. Acetone was purchased from E. Merck (Germany) while prednisolone and oxazolone were purchased from Sigma (U.S.A.). Animals Female CBA mice, purchased from Iffa-Credo (France), were used for all of the experiments described in this report. Animals were 4 weeks of age at the time of arrival and were allowed 10 days acclimatization before beginning the study. The temperature of the room where the mice were housed was maintained at 2 0 - 2 2 ° C . Mice were allowed free access to water and a standard mouse diet (Nafag, Switzerland) throughout the study. Early initiation phase studies For these studies groups of four mice were sensitized (day 1) with a 3O7o (w/v) solution of oxazolone in acetone by application of 130 ~1 to the abdomen and thorax under standard conditions. Forty hours later (day 3) the initial ear thickness was determined using a micrometer. Immediately thereafter the oxazolone challenge was applied to the dorsal surface of both ears as 5 tA of a 0.6% (w/v) solution of oxazolone in olive oil. Ear thickness was then determined 2 h later. Delayed hypersensitivity studies For these studies groups of four mice were sensitized (day 1) with a 3% (w/v) solution of oxazolone in acetone by application of 50 ~1 to the abdomen under standard conditions. Six days later (day 7) the initial ear thickness was determined. Immediately thereafter the oxazolone challenge was applied to the dorsal surface of both ears as 5/~1 of a 0.6% (w/v) solution of oxazolone in olive oil. Ear

LCB 2183 Inhibits Inflammation Table 1. Ear-swelling response in relation to time after challenge in early initiation phase studies Time after challenge (h) 1 2 4 24

8 -

Specific increment in ear thickness (cm × 10 3) 4 4 4 4

1.50 5.00 -0.25 1.13

+ 0.38* -+ 0.84* 4- 1.05 4- 0.54

N.S. T

N.S.

s/l, / / / i

.

.

.

.

.

:0.005

.

4 ~/~//

Data are mean _+ S.E.M. *Indicates that ear-swelling is statistically significant (paired t-test). thickness was t h e n d e t e r m i n e d 24 h (and in some experiments 30 h or 48 h) later.

677

~//

2p <0.--005

000,

&

y//

........ / / / /

A dministration o f test products / / / /

F o r a d m i n i s t r a t i o n by intragastric gavage, test p r o d u c t s were dissolved in distilled water immediately before use. In some experiments the test p r o d u c t s were a d m i n i s t e r e d by i n t r a p e r i t o n e a l injection (because nedocrornil s o d i u m a n d s o d i u m cromoglycate are very poorly a b s o r b e d f r o m the g a s t r o i n t e s t i n a l tract), in this case they were dissolved in p h o s p h a t e b u f f e r e d saline i m m e d i a t e l y b e f o r e use. C o n t r o l mice were treated with vehicle alone. Solutions were p r e p a r e d such t h a t the indicated doses were a d m i n i s t e r e d in a v o l u m e o f 4 ml/kg. In all of the early initiation phase studies test p r o d u c t s were a d m i n i s t e r e d o n 4 consecutive days (day 0 to day 3) b e g i n n i n g 1 day before the sensitization a n d with the last dose being a d m i n i s t e r e d 1 h before the challenge. Except w h e n otherwise indicated, test p r o d u c t s were a d m i n i s t e r e d by intragastric gavage. In the delayed hypersensitivity studies the exact a d m i n i s t r a t i o n schedule varied d e p e n d i n g o n the objective o f each experiment. Details are p r o v i d e d in the text.

Treatment o f the data In all experiments a g r o u p o f four oxazolone-naive mice were challenged with o x a z o l o n e in order to d e t e r m i n e the non-specific ear-swelling response. The i n c r e m e n t in ear thickness was d e t e r m i n e d for each a n i m a l as a n average o f the two ears. T h e specific i n c r e m e n t was t h e n calculated by s u b t r a c t i n g the non-specific ear-swelling o b t a i n e d with the o x a z o l o n e - n a i v e mice f r o m the ear swelling o b t a i n e d with sensitized mice (non-specific ear-swelling was routinely less t h a n 2 x 10 _3 cm). T h e m e a n a n d s t a n d a r d error o f the m e a n were t h e n calculated for

o ~///

H20 0.8 4 20 1 5 25 Prednisolone mg/kg P.O. LCB 2183 mg/kg P.O.

Fig. 1. Inhibition of the early initiation phase of contact sensitivity by oral administration of prednisolone or LCB 2183. Groups of four CBA mice were treated with test products as indicated on 4 consecutive days (days 0 - 3). A sensitizing dose of oxazolone was applied to the abdomen and thorax on day 1. Forty hours later (day 3), and 1 h after the last administration of test product, a challenge dose of oxazolone was applied to the dorsal surface of each ear. The increment in ear thickness was determined for each animal 2 h later (ear thickness at 2 h minus initial ear thickness). Non-specific ear-swelling (0.25 _+ 0.48), determined with a group of oxazolone naive mice, was then subtracted. Data are mean _+ S.E.M. The relationship between log dose and inhibition of ear-swelling is statistically significant with both drugs. each g r o u p o f mice. A two-tailed t-test was t h e n used for statistical c o m p a r i s o n o f the means. Values o f P less t h a n 0.05 were considered to indicate statistically significant differences.

RESULTS

Early initiation phase studies T h e transient n a t u r e o f the early initiation phase is illustrated in T a b l e 1. A statistically significant earswelling response was observed at 1 h, was o p t i m a l at 2 h a n d was n o longer observed at 4 h. No late p h a s e response (at 24 h) was observed u n d e r the conditions used for these experiments. The 2 h ear-swelling response was a b r o g a t e d by intragastric a d m i n i s t r a t i o n o f p r e d n i s o l o n e at a dose level o f 20 m g / k g [Fig. 1 a n d Table 2(b)]. Lower

678

N. MURRAYet al.

Table 2. The effect of drugs on the 2 h ear-swelling response

(a)

(b)

Treatment group

n

Specific increment in ear thickness (cm x 10 -3)

Percent inhibition of ear swelling

Control

4

4.875 + 0.72

LCB 2183 (p.o.) 11.1 mg/kg

4

2.000 ± 1.24

59%

LCB 2183 (p.o.) 33.3 mg/kg

4

1.875 ± 0.43*

61%

LCB 2183 (p.o.) 100 mg/kg

4

2.000 ± 0.68*

59%

Control

4

4.500 ± 0.31

m

Nedocromil sodium (i.p.) 25 mg/kg

4

3.375 ± 0.20*

25°7o

Sodium cromoglycate (i.p.) 25 mg/kg

4

5.125 + 0.78

_ 1407o

Prednisolone (p.o.) 20 mg/kg

4

0.250 ± 0.24*

94%

Data are mean _+ S.E.M. *Indicates that means are significantly different from control. Table 3. Ear-swelling response in relation to time after challenge in late hypersensitivity studies Time after challenge (h)

n

Specific increment in ear thickness (cm x 10 ~)

2 24 30

4 4 4

1.75 ± 0.43* 7.38 _+ 0.13" 5.75 _+ 0.43*

Data are mean ± S.E.M. *Indicates that ear-swelling is statistically significant (paired t-test). doses of prednisolone were less effective and indeed a statistically significant straight line relationship between log dose and inhibition of ear-swelling was observed (r = 0.88, P<0.05; Fig. 1). The 2 h response was partially inhibited by intragastric administration of LCB 2183 at a dose level of 25 m g / k g . A statistically significant straight line relationship between log dose of LCB 2183 and inhibition of ear-swelling was also observed (r = 0.62, P<0.05; Fig. 1). Further increasing the dose of LCB 2183 did not, however, further increase the degree of inhibition [Table 2(a)]. The standard anti-allergic c o m p o u n d , nedocromil sodium, inhibited the 2-h ear-swelling response to a limited extent when administered by the intraperitoneal route at a dose level of 25 m g / k g . Under the same conditions sodium cromoglycate had no

effect [Table 2(b)]. A repeat experiment confirmed that sodium cromoglycate was inactive at 100 m g / k g and showed that the degree of inhibition induced by nedocromil sodium was no greater with 100 than with 25 m g / k g (not shown).

Delayed hypersensitivity studies Preliminary studies revealed that under the conditions described for these experiments optimal ear-swelling responses were observed 2 4 - 3 0 h after oxazolone challenge. Two hour ear-swelling was much less marked than in the above experiments; it was, nonetheless, statistically significant (Table 3). The early initiation phase studies revealed that LCB 2183 could significantly inhibit the 2 h response when it was administered on 4 consecutive days at a dose level of 25 m g / k g . However, it was not clear whether the inhibitory effect of LCB 2183 was on the afferent (induction of sensitization) or the efferent (effector response) limb of the hypersensitivity response; this was because the delay between sensitization and challenge with oxazolone was only 40 h. Thus, both the sensitization (day 1) and the challenge (day 3) were applied during the period of drug treatment (day 0 to day 3). In the delayed hypersensitivity experiments the period between the sensitization (day 1) and the challenge (day 7) was 6 days. We therefore had the opportunity to study whether the afferent or the

679

LCB 2183 Inhibits Inflammation Table 4. The effect of drug administration around the time of sensitization (day 1) or challenge (day 7) on the 24 h ear-swelling response

n

Specific increment in ear thickness (cm × 10 3)

Percent inhibition

Control (H20 days 0 - 7 )

4

7.375 _ 0.125

--

LCB 2183 days 0 - 3 (25 mg/kg p.o.) + H20 d a y s 4 - 7

4

6.375 _+0.875

13°70

LCB 2183 days 4 - 7 (25 mg/kg p.o.) + H20 d a y s 0 - 3

4

3.000 _ 0.66*

59o/0

Prednisolone days 0 - 3 (20 mg/kg p.o.) + HzOdays4-7

4

3.375 _+0.31"

54o/0

Prednisolone days 4 - 7 (20 mg/kg p.o.) + H20 days 0 - 3

4

0.625 _+0.47*

91%

Treatment group

Data are mean _ S.E.M. *Indicates that means are significantly different from control.

efferent limb of the response was inhibited by LCB 2183. The drug was administered in the first experiment (Table 4) by intragastric gavage on 4 consecutive days at a dose level of 25 mg/kg, separate groups of mice received the active treatment on days 0 - 3 or on days 4 - 7 . The last administration of product on day 7 was 1 h before challenge with oxazolone. It can be seen that LCB 2183 induced statistically significant inhibition of the 24 h ear-swelling response only when administered for the 4 days immediately before the challenge. Administration for 4 days around the time of sensitization was ineffective. With prednisolone, the ear-swelling response was abrogated by administration immediately before challenge and strongly inhibited by administration around the time of sensitization. Similar results were obtained with the 30 h ear-swelling response (data not shown). Furthermore, with both LCB 2183 and prednisolone the inhibition of the 24 h ear-swelling response was dose dependent (LCB 2183 r - - 0 . 6 7 , P<0.05; prednisolone r -- 0.92, P<0.05; Fig. 2). In order to determine whether administration of LCB 2183 on 4 consecutive days was necessary for full activity we compared administration on 1, 2, 3 or 4 occasions as shown in Table 5. It can be seen that a single oral dose of the drug 1 h before challenge induced statistically significant inhibition of the 24 h ear-swelling response. The degree of inhibition was

significantly enhanced by an additional administration 24 h previously and still further enhanced by administration 48 and 24 h previously. An additional administration 72 h previously produced no further enhancement of the response. Full activity was therefore observed when LCB 2183 was administered on 3 consecutive days. The standard anti-allergic drugs nedocromil sodium and sodium cromoglycate were then tested for activity in this model. Administration of 25 m g / k g by the intraperitoneal route on 3 consecutive days (days 5 to 7) was found to be ineffective with both of these anti-allergic compounds. In contrast, LCB 2183 induced statistically significant inhibition of the 24 h ear swelling response when administered at 25 mg/kg on 3 consecutive days either by the intraperitoneal route or by intragastric gavage (Table 6), similar results were obtained at 30 and 48 h (data not shown). A repeat experiment confirmed that neither nedocromil sodium nor sodium cromoglycate were active in the late phase when administered at 100 mg/kg by the intraperitoneal route on 4 consecutive days (not shown). DISCUSSION In order to study drug effects on the early and late components of contact sensitivity we have used two separate protocols. In the first, the early initiation

680

N. MURRAYet al.

5

N.S.N.S. ~ 4 ,/// ~ E 3

2p <0.05

9 .E

&y/ 2

2p <0.02

//")

K

,

////

,'/// /,

o

Y//

H20 0.8 4 20 1 5 25 Prednisolone mg/kg P.O. LCB 2183 mg/kg P.O

Fig. 2. Inhibition of the delayed response of contact sensitivity by oral administration of prednisolone or LCB 2183. Groups of four CBA mice were treated with test product as indicated on 4 consecutive days (days 4-7). A sensitizing dose of oxazolone was applied to the abdomen on day 1. Six days later (day 7) and 1 h after the last administration of test product a challenge dose of oxazolone was applied to the dorsal surface of each ear. The increment in ear thickness was determined for each animal 24 h later (ear thickness at 24 h minus initial ear thickness). Non-specific ear-swelling (1.375 ± 0.125), determined with a group of oxazolone-naive mice, was then subtracted. Data are mean _+ S.E.M. The relationship between log dose and inhibition of ear-swelling is statistically significant with both drugs. phase is very pronounced but the late phase does not occur because the delay between oxazolone sensitization and challenge (40 h) is too short for effector T-cell clonal selection, expansion and maturation (Van Loveren, Meade & Askenase, 1983). In the second, the late phase is potentiated at the expense of the early initiation phase. These protocols were developed on the basis of published data (Van Loveren, Meade & Askenase, 1983; Van Loveren et al., 1984) and the expected and observed results correspond perfectly. The early initiation phase of contact sensitivity is associated with relatively selective liberation of serotonin from mast cells (Van Loveren, KraeuterKops & Askenase, 1984), liberation of prostaglandin E2 and leukotrienes B4 and C4 (Meurer, Opas & Humes, 1988), deposition of fibrin (Mekori & Galli, 1990) and increased vascular permeability (Askenase, Rosenstein & Ptak, 1983). In our experiments,

the ear-swelling response associated with the early component of contact sensitivity was abrogated by oral prednisolone, inhibited to about 50°7o by oral LCB 2183 and inhibited to a lesser extent by nedocromil sodium, which was administered by the i.p. route since it is poorly absorbed after intragastric administration. Sodium cromoglycate was also tested by the i.p. route, since it too is poorly absorbed from the gastrointestinal tract, but it was ineffective. In a DTH-like model in the mouse lung (Garssen, Nijkamp, Wagenaar, Zwart, Askenase & Van Loveren, 1989) the early component not only initiates T-cell recruitment leading to inflammation but also provokes airway narrowing and hyperreactivity (Garssen, Nijkamp, Van der Vliet & Van Loveren, 1991). Therefore, one could speculate that drugs which inhibit the early initiation phase could also inhibit the development of increased airway resistance and hyper-reactivity in this lung model. Indeed, this has apparently been shown with corticosteroids and nedocromil sodium (Askenase, 1992) and we now intend to determine whether or not LCB 2183 inhibits the early component and consequent airway changes in this lung model. The delayed response to hapten challenge depends on the occurrence of the early initiation phase (Van Loveren & Askenase, 1984). In the present study we have examined whether drugs which inhibit the early phase also inhibit the delayed response, as predicted. Both prednisolone and LCB 2183 inhibited the efferent limb of the late inflammatory response, possibly as a direct consequence of early phase inhibition, but drug effects on the late acting inflammatory cells cannot be excluded. Unexpectedly, nedocromil sodium, which had a limited inhibitory effect on the initiation phase, did not significantly influence the delayed inflammatory response. The reason for this is unclear at the present time. Like nedocromil sodium and sodium cromoglycare, LCB 2183 is known to inhibit IgE-mediated responses, such as passive cutaneous anaphylaxis in animals (unpublished data). However, as shown here, LCB 2183 differs from these products in its ability to inhibit the late inflammatory component of contact sensitivity. LCB 2183 may therefore have more potential for anti-inflammatory effects and, indeed, in the model used here this product resembled prednisolone more than nedocromil sodium. The fact that LCB 2183 is active after intragastric administration constitutes another important difference between it and the standard anti-allergic drugs. Further study in the related

681

LCB 2183 Inhibits Inflammation Table 5. The relationship between the number of days of administration of LCB 2183 (25 mg/kg p.o.) and the degree of inhibition of the 24 h ear-swelling response Number of days treatment with active drug

n

Control (H20 days 4 - 7 )

0

4

6.750 _+ 0.14

--

LCB 2183 (day 7) + H20 (days 4 - 6 )

1

4

5.250 _+ 0.25*

22070

LCB 2183 (days 6+7) + HzO (days 4 + 5)

2

4

3.625 _+ 0.24*

46%

LCB 2183 (days 5 - 7 ) + HzO (day 4)

3

4

2.625 + 0.125"

61%

LCB 2183 (days 4 - 7 )

4

4

2.625 _ 0.125"

61070

Treatment group

Specific increment in ear thickness Percent (cm x 10 -3) inhibition

Data are mean _+ S.E.M. *Indicates that means are significantly different from control. Table 6. The effect of drug administration during 3 consecutive days before challenge on the 24 h ear-swelling response

n

Specific increment in ear thickness (cm x 10 -3)

Percent inhibition

Control (PBS days 5 - 7)

4

5.000 +_ 0.51

--

Nedocromil sodium (25 mg/kg i.p.)

4

5.500 _ 0.24

- 10%0

Sodium cromoglycate (25 mg/kg i.p.)

4

4.750 _+ 0.24

5%

LCB 2183 (25 mg/kg i.p.)

4

2.500 _ 0.55*

500/0

LCB 2183 (25 mg/kg p.o.)

4

2.875 _+ 0.14"

50%

Treatment group

Data are mean__. S.E.M. *Indicates that means are significantly different from control. m o u s e lung m o d e l (Garssen, N i j k a m p , W a g e n a a r , Zwart, A s k e n a s e & Van Loveren, 1989; Garssen, N i j k a m p , Van der Vliet & Van Loveren, 1991) will reveal whether or not LCB 2183 is able to inhibit i n f l a m m a t i o n as well as the increased airway

resistance and bronchial hyper-reactivity. In addition, phase II clinical studies will determine whether LCB 2183 has true potential as an antia s t h m a drug.

682

N. MURRAY et al. REFERENCES

ALEXANDER, A. G., BARNES, N. C. & KAY, A. B. (1992). Trial of cyclosporin in corticosteroid-dependent chronic severe asthma. Lancet, 339, 3 2 4 - 3 2 8 . ASKENASE, P. W. (1992). Delayed-type hypersensitivity recruitment of T-cell subsets via antigen-specific non lgE factors or IgE antibodies: relevance to asthma, a u t o i m m u n i t y and i m m u n e responses to tumors and parasites. In The Regulation and Functional Significance o f T-cell Subsets (ed. R. C o f f m a n n ) , pp. 1 - 77. Karger, Basel. ASKENASE, P. W., BURSZTAJN, S., GERSHON, M. D. & GERSHON, R. K. (1980). T-cell-dependent mast cell degranulation and release of serotonin in murine delayed-type hypersensitivity. J. exp. Med., 152, 1 3 5 8 - 1374. ASKENASE, P. W., ROSENSTEIN, R. W. & PTAK, W. (1983). T-cells produce in antigen binding factor with in vivo activity analogous to IgE antibody. J. exp. Med., 157, 8 6 2 - 8 7 3 . BARNES, P. J. (1989). Overview of current therapy. In New Drugs for Asthma (ed. P. J. Barnes), pp. 1 - 1 1 . IBC Technical Services, London. BROWN, P. H., CROMPTON,G. K. & GREENING, A. P. (1991). P r o i u f l a m m a t o r y cytokines in acute asthma. Lancet, 338, 590 - 593. CORRIGAN, C. J., HARTNELL, A. & KAY, A. B. (1988). T-lymphocyte activation in acute severe asthma. Lancet, i, 1 1 2 9 - 1132. CRUZ, P. D., TIGELAAR, R. E. & BERGSTRESSER, P. R. (1990). Langerhans cells that migrate to skin after intravenous infusion regulate the induction of contact hypersensitivity. J. Immun., 144, 2 4 8 6 - 2 4 9 2 . FLOOD, P., PTAK, W. & GREEN, D. R. (1986). Mechanism of action of a T-suppressor factor (TsF) in contact sensitivity: The T-cell target for TsF activity in adoptive transfer of immunity is not the effector cell. J. hnmun., 137, 1 8 2 9 - 1835. GARSSEN, J., NIJKAMP, F. P., VAN DER VLIET, H. & VAN LOVEREN, H. (1991). T-cell mediated induction of airway hyperreactivity in mice. Am. Rev. resp. Dis., 144, 9 3 1 - 9 3 8 . GARSSEN, J., N1JKAMP, F. P. WAGENAAR, S. S., ZWART, A., ASKENASE, P. W. & VAN LOVEREN, H. (1989). Regulation of delayed-type hypersensitivity-like responses in the m o u s e lung, determined with histological procedures: serotonin, T-cell suppressor-inducer factor and high antigen dose tolerance regulate the magnitude of T-cell dependent inflammatory reactions. Immunology, 68, 5 1 - 58. GASPARI, A. A. & KATZ, S. I. (1991). Induction of in vivo hyporesponsiveness to contact allergens by hapten-modified Ia + keratinocytes. J. Immun., 147, 4155 - 4161. GAUTAM, S. C., CH1KKALA, N. F. & HAMILTON, T. A. (1992). Anti-inflammatory action of IL4. Negative regulation of contact sensitivity to trinitrochlorobenzene. J. Immun., 148, 1411 - 1415. GIROLOMONI, G. & T1GELAAR, R. E. (1990). Capsaicin-sensitive primary sensory neurons are potent modulators of murine delayed-type hypersensitivity reactions. 145, 1105 - 1112. GOCINSKI, B. L. & TIGELAAR, R. E. (1990). Roles of CD~ + and C D 8 T-cells in murine contact sensitivity revealed by in vivo monoclonal antibody depletion. J. Immun., 144, 4121 - 4 1 2 8 . HERZOG, W.-R., FERRERI, N.R., PTAK, W. & ASKENASE, P. W. (1989). The DTH-initiating Thy-1 ~ cell is double negative (CD4 , C D s ) and CD3 and expresses IL-3 receptors, but no 1L-2 receptors. J. Immun., 143, 3 1 2 5 - 3 1 3 3 . HERZOG, W.-R., MEADE, R., PETTINICCHI, A., PTAK, W. & ASKENASE, P. W. (1989). Nude mice produce a T-cell-derived antigen-binding factor that mediates the early c o m p o n e n t of delayed-type hypersensitivity. J. Immun., 142, 1803 - 1812. HERZOG, W.-R., MILLET, J., FERRER1, N. R., RAMABHADRAN,R., SCHREURS, J. & ASKENASE, P. W. (1990). An antigenspecific DTH-initiating cell clone. Y. Immun., 144, 3 6 6 7 - 3 6 7 6 . HERZOG, W.-R., PTAK, W. & ASKENASE, P. W. (1990). Suppression and contrasuppression in athymic nude mice: nude mice produce the antigen-specific c o m p o n e n t of a T-suppressor factor that inhibits the late 24 h phase of DTH, but does not generate suppression nor contrasuppression of the early initiating phase of DTH. Cell. Immun., 127, 1 3 0 - 145. MATSUSHIMA,G. K. & STOHLMAN,S. A. (1991). Distinct subsets of accessory cells activate Thy-1 + triple negative (CD3 , CD4 , CDs-) cells and Th-I delayed-type hypersensitivity effector T-ceUs. J. Immun., 146, 3 3 2 2 - 3 3 3 1 . MEADE, R., VAN LOVEREN, H., PARMENTIER, H., IVERSON, G. M. & ASKENASE, P. W. (1988). The antigen-binding T-cell factor P C L - F sensitizes mast cells for in vitro release of serotonin. J. Immun., 141, 2 7 0 4 - 2 7 1 3 . MEKORI, Y. A. & GALLI, S. J. (1990). Fibrin deposition occurs at both early and late intervals of IgE-dependent or contact sensitivity reactions elicited in mouse skin. J. Immun., 145, 3 7 1 9 - 3 7 2 7 . MEURER, R., OPAS, E. E. & HUMES, J. L. (1988). Effects o f cyclooxygenase and lipoxygenase inhibitors on inflammation associated with oxazolone-induced delayed hypersensitivity. Biochem. Pharmac., 37, 3 5 1 1 - 3514.

LCB 2183 Inhibits Inflammation

683

PTAK, W., ASKENASE,P. W., ROSENSTEIN, R. W. & GERSHON, R. K. (1982). Transfer of an antigen-specific immediate hypersensitivity-like reaction with an antigen-binding factor produced by T-cells. Proc. natn. Acad. Sci. U.S.A., 79, 1969 - 1973. PTAK, W., GEBA, G. P. & ASKENASE,P. W. (1991). Initiation of delayed-type hypersensitivity by low doses of monoclonal IgE antibody. J. Immun., 146, 3929-3936. PTAK, W., GERSHON, R. K., ROSENSTE1N,R. W., MURRAY,J. H. & CONE, R. E. (1983). Purification and characterization of TNP-specific immunoregulatory molecules produced by T-cells sensitized by Picrylchloride. J. lmmun., 131, 2859-2863. PTAK, W., HERZOG, W.-R. & ASKENASE,P. W. (1991). Delayed-type hypersensitivity initiation by early-acting cells that are antigen mismatched or MHC incompatible with late-acting, delayed-type hypersensitivity effector T-cells. J. Irnmun., 146, 469-475. SULLIVAN, S., BERGSTRESSER, P. R., TIGELAAR, R. E. & STREILEIN, J. W. (1986). Induction and regulation of contact hypersensitivity by resident, bone marrow-derived, dendritic epidermal cells: Langerhans cells and Thy-I + epidermal cells. J. Immun., 137, 2460-2467. VAN LOVEREN,H. & ASKENASE,P. W. (1984). Delayed-type hypersensitivity is mediated by a sequence of two different Tcell activities. J. Immun., 133, 2397-2401. VAN LOVEREN, H., DE WEGER, R. A., GARSSEN,J., Los, G. & ASKENASE,P. W. (1989). Impairment of allograft tumor immunity by isotype-like suppression of antigen-specific T-cell factors. Transplantation, 47, 504-512. VAN LOVEREN, H., KATO, K., MEADE, R., GREEN, D. R., HOROWITZ, M., PTAK, W. & ASKENASE, P. W. (1984). Characterization of two different Ly-P cell populations that mediate delayed-type hypersensitivity. J. Immun., 133, 2402 - 2411. VAN LOVEREN, H., KRAEUTER-KOPS,S. & ASKENASE,P. W. (1984). Different mechanisms of release of vasoactive amines by mast cells occur in T-cell-dependent compared to IgE-dependent cutaneous hypersensitivity responses. Eur. J. Immun., 14, 40-47. VAN LOVEREN,H., MEADE,R. & ASKENASE,P. W. (1983). An early component of delayed-type hypersensitivity mediated by T-cells and mast cells. J. exp. Med., 157, 1604- 1617. VAN LOVEREN, H., RATZLAFF,R. E., KATO, K., MEADE, R., FERGUSON, T. A., IVERSON, G. M., JANEWAY,C. A. & ASKENASE, P. W. (1986). Immune serum from mice contact-sensitized with picryl chloride contains an antigenspecific T-cell factor that transfers immediate cutaneous reactivity. Eur. J. Immun., 16, 1203 - 1208. WALKER, C., VIRCHOW, J.-C., BRUIJNZEEL, P. L. B. & BLASER, K. (1991). T-cell subsets and their soluble products regulate eosinophilia in allergic and nonallergic asthma. J. Immun., 146, 1829- 1835. WELSH, E. A. & KRXPKE,M. L. (1990). Murine Thy-1 ÷ dendritic epidermal cells induce immunological tolerance in vivo, J. Immun., 144, 883- 891.