Immunobiol., vol. 186, pp. 351-361 (1992) Physiology Unit, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
Adjuvant Arthritis Pretreatment with Type II Collagen and Mycobacterium butyricum ANGELS FRANCH, SALVADOR CASSANY, CRISTINA CASTELLOTE, and MARGARIDA CASTELL Received December 9, 1991 . Accepted in Revised Form June 23, 1992
Abstract A treatment previous to adjuvant arthritis induction has been performed with type II collagen (ell) or Mycobacterium butyricum (Mb), which is the inducer of the pathology. Pretreatment was administered in two different ways: a) subcutaneously or intradermally 14 days before arthritis induction, and b) intravenously 3 days before induction. In order to relate the change in inflammation to the corresponding antigen immune response, serum antibodies and delayed type hypersensitivity (DTH) against ell or Mb were studied. Pretreatment with s.c. ell 14 days before induction produced slight protection against arthritis and significantly delayed its onset; systemic inflammation showed good positive correlation with anti-ell antibodies. The ell administered i.v. 3 days before arthritic challenge did not significantly modify the inflammatory process. The use of i.d. subarthritogenic doses of Mb 14 days before induction protected a high percentage of the animals from the posterior arthritic challenge; this protection was accompanied by high anti-Mb antibody titers and DTH reaction. When Mb was given i.v. 3 days before induction, a partial protection of inflammation was observed; arthritis was milder and its onset was delayed. These changes were accompanied by reduced humoral and cellular response to Mb.
Introduction Adjuvant arthritis (A A) is an animal model of polyarthritis, which is induced by subplantar or intradermal injection of a suspension of certain mycobacteria. The etiology of this pathology has not been established, although it appears to be mediated by immunological mechanisms, because the disease can be transferred by cell populations which are specific for mycobacteria (1-3). Autoimmune etiology has received support from several studies (4-6) and VAN EDEN et al. (7) have suggested that the primary auto antigen is a 65- KDa heat-shock protein. In AA a cellular immune response to CII and anti-collagen antibody activity has been reported (5, 8). Abbreviations: AA = adjuvant arthritis; AI = arthritic index; BSA = bovine serum albumin; II collagen; DMAB = 3-dimethylaminobenzoic acid; DTH = delayed type hypersensitivity; ELISA = enzyme-linked immunosorbent assay; i.d. = intradermal; IFA = incomplete Freund's adjuvant; i. v. = intravenous; Mb = Mycobacterium butyricum; MBTH = 3methyl-2-benzothiazolinone-hydrazone-hydrochloride; PBS = phosphate-buffered saline; PBS- T-BSA = PBS containing 0.5 % Tween and 1 % BSA; s.c. = subcutaneous
en = type
352 . A. FRANCH, S. CASSANY, C. CASTELLOTE, and M. CASTELL
On the other hand, the inducer of AA has acquired importance because there is evidence implicating mycobacterial infections as agents capable of initiating autoimmunity. In patients suffering from pulmonary tuberculosis, antibodies against nuclear components, rheumatoid factor and antiDNA auto-antibodies have been described (9, 10). Furthermore, in arthritic patients anti-M. tuberculosis and anti-heat shock protein antibodies have been detected (11). These findings suggest that mycobacterial infection could be responsible for auto-antibody development, owing to molecular mimicry between mycobacterial structure and some endogenous components. In order to study the roles of mycobacteria and collagen in AA, we have attempted to modify adjuvant arthritis development through immunization with type II collagen (s.c.) and subarthritogenic doses of M. butyricum (i.d.), 14 days before arthritis induction. Intravenous pretreatment with the same agents 3 days before arthritic challenge was also administered. Serum antibodies and DTH against ell or Mb have been determined in order to elucidate the potential pathogenetic role of immune responses to collagen type II and disease-inducing mycobacteria.
Material and Methods Animals Outbred female Wi star rats (Interfauna Iberica S.A., Barcelona, Spain) weighing 200-220 g were housed in groups of 4 to 6 in plastic cages with food and water ad libitum. The temperature was regulated and kept within the range 22 ± 2 0c. The animals were maintained under a 12 h light/12 h darkness cycle.
Collagen and mycobacteria Soluble-form chicken type II collagen was obtained from Genzyme Corporation, Boston, MA, USA. Heat killed Mycobacterium butyricum was purchased from Difco Laboratories, Detroit, MI, USA.
Pretreatment protocol CII or Mb were administered following the protocol: - One group of 10 rats was injected s.c. with 1 ml of an emulsion prepared with IFA and CII in PBS (final concentration: 500 [.lg/ml), at several sites on the back on day -14 before induction of AA. - One group of 12 rats was injected i.v. in the tail with 1 mg of CII in 1 ml PBS, on day -3 prior to induction. - One group of 10 rats was injected i.d. with 50 [.lg Mb in 0.1 ml of mineral oil, at the base of the tail on day -14 before induction. - One group of 10 rats was injected i.v. with 2 mg of Mb in 1 ml PBS in the tail, on day -3 before induction of AA. Mb was prepared from Mb pulverized in a mortar and sonicated in PBS. In addition to the four pretreated groups, eight more groups were established: an arthritic group and a healthy group without pretreatment; four healthy groups which received the above-mentioned pretreatments and finally, two arthritic groups pretreated with BSA as a protein control. In these latter groups, BSA was administered s.c. 14 days before induction or i.v. three days before arthritic challenge; the protocols were the same as those indicated for CII pretreatments.
Adjuvant Arthritis Pretreatment·
Induction and assessment of arthritis Rats were intradermally injected at the base of the tail with 500 [tg Mb in 0.1 ml of mineral oil. Healthy groups received 0.1 ml of mineral oil under the same conditions. The lesions of the four paws were each graded from 0 to 4 according to erythema and edema (measured with a mercury plethysmometer) of the periarticular tissue. The arthritic index (AI) for each animal was the sum of the score for each of the four paws, the maximum value of AI was 16. Evaluation of arthritis was carried out weekly from day 0 to 35 and daily between day 11 and day 15. An AI below 2 was considered negative, arthritis was considered mild if AI was between 2-5, moderate between 6-9 and severe between 10-16.
Samples Blood samples were obtained weekly from each animal. After ether anaesthesia, blood was collected from rats by retroorbital bleeding. Serum was stored at -20°C until use.
Anti-elI and anti-Mb antibody quantification An enzyme-linked immunosorbent assay (ELISA) was performed. Polystyrene microELISA plates of 96 wells (Dynatech, Switzerland) were incubated overnight with ClI (10 [tgl ml, at 4°C) or a soluble protein fraction of Mb (3 [tg/ml, at room temperature, obtained as described in 12) in PBS. Active sites on the plastic surface were then blocked (1 h at room temperature with PBS containing 1 % BSA). The standard and the samples were diluted in PBS-Tween 0.5% containing 1 % BSA (PBS-T-BSA) and were incubated for 3h at room temperature. For anti-ClI antibody determination, samples were diluted 1/800 (s.c. ClI immunized animals) or 1120 (arthritic rats and i.v. ClI administered animals). To study antiMb antibodies, all samples were diluted 1/1000. After washing, anti-rat immunoglobulins conjugated to horseradish peroxidase (Dako) were added. Color was developed with the substrate DMAB-MBTH (13) and measured at 600 nm using a Titertek Uniskan (Flow Lab, Helsinki, Finland). The absorbance data were analyzed by the ELISA CALC computer program (12) and results are expressed in concentration units, according to the arbitrary units attributed to a pool standard.
Delayed type hypersensitivity reaction (DTH) DTH reactions were measured on day 35 after induction. Rats were intradermally injected in the left ear with 75 [tg of ClI or Mb in 50 [tl of PBS. The right ear of each animal received an equivalent volume of PBS. After 48 h, the thickness of each ear was measured with a micrometer. The ratio of thickness of the CII- or Mb-injected ear to that of the PBS-injected ear served as an index of DTH response (LE/RE).
Statistical analysis Student's t-test for two means was applied to establish the level of significance of antibody and DTH responses. For arthritis onset and arthritic index, the Mann-Whitney's U test was used. When the data represent the results obtained throughout the studied period, the probability was calculated using the areas under the curve. The X 2 test was used to calculate statistical significance for the incidence of the arthritis.
Effect of pretreatments on systemic inflammation The incidence, severity and onset of AA from the arthritic control group and arthritic pretreated groups are shown in Table 1. The inflammation degree (arthritic index) of these groups is shown in Figure 1. Those groups pretreated s.c. or i.v. with BSA developed an inflammation similar to the
(1 ) 13.14 ± 0.99':' (7)
BSA, via s.c. on day -14
BSA, via i.v. on day-3
':. p < 0.05, ,',:. p < 0.01, significantly different from arthritic control group
Mb, via i.v. On day-3
Mb, via i.d. On day -14
12.25 ± 0.48 (4)
12.40 ± 0.40 (5)
13.09 ± 0.59 (11)
>14.62 ± 1.24':' (8)