Adjuvant arthritis
Table
3 reports the clinical scores of AA. Prevention of paw swelling after treatment with UF-S and UF-f/t was statistically significant (p < 0.01) compared with the control groups. These results showed that active unknown epitopes would be released in UF-S even during 3 h of culture (in the absence of Rifamycin), following a partial spontaneous cytolysis. On the contrary, AA was not inhibited in rats treated with UF-R and Rifamycin alone (Table
3) and also by the UF-sf from patients with arthrosis (Table
4). Arthrosis is commonly accepted as the normal control for RA.
Table 3
Modulation of adjuvant arthritis development in rats by administration of ultrafiltrates obtained from PBMCs of RA patients
11 | 00000 00233 | 0 | 00000 11246 | 0.5 | 00122 22223 | 2 | 00001 11222 | 1 | 00011 22245 | 1.5 |
15 | 00012 24457 | 2 | 02222 33357 | 2.5 | 12223 33344 | 3 | 02333 33444 | 3 | 24445 55666 | 3 |
19 | 11244 45588 | 4 | 02344 45557 | 4 | 24555 66788 | 5.5 | 25555 66788 | 5.5 | 46666 67777 | 5.5 |
23 | 13333 55788 | 4 | 03344 55567 | 4.5 | 24555 66888 | 5.5 | 33344 56668 | 4.5 | 45555 66678 | 4.5 |
27 | 12223 36778 | 3 | 02344 44457 | 4 | 23455 55888 | 5 | 33334 55668 | 4.5 | 44445 55668 | 4.5 |
Table 4
Protection of rats from adjuvant arthritis by administration of synovial fluid (UF-sf)
2.37 ± 1.60º | 5.9 ± 1.01^ | 6.7 ± 095^ | 7.5 ± 0.92^ |
The administration of long-lasting (more than a month) RA synovial fluid, UF-sf, induced a significant reduction of the severity of subsequent arthritis in rats (p < 0.01) while SF from recent RA effusion (5–10 days after a previous complete extraction) was ineffective (Table
4). The ineffectiveness of the recent fluid is probably due to the gradual accumulation over time of immunoactive epitopes from lysis of proliferating synoviocites and deeper mononuclear cells in inflamed synovium. The control saline treated animals had a severe arthritis, as demonstrated by the high clinical scores (Table
4).
In conclusion, longstanding synovial fluid, UF-sf, the supernatant of PBMC cultures, UF-S, and Ultrafiltrate of PBMCs fragmented with freeze/thawing, UF-f/t, all from subjects with RA, are able to modulate the immune mechanism implicated in AA and to stimulate protective immunity.
In Table
5, we summarize the results of autologous immunotherapy in RA patients and the protection profile of the human ultrafiltrates administered in rats with oncoming arthritis.
Table 5
Autologous immunotherapy of RA with ultrafiltrates derived from PBMCs and synovial fluids. The protection profile in AA is included
Prepared from acellular synovial fluid | UF-sf | Active | Active |
Prepared by freeze/thawing of PBMCs | UF-f/t | Active | Active |
Prepared from supernatant of PBMCs culture | UF-S | ND | Active |
Prepared by lysing PBMCs with rifamycin SV | UF-R | Active | Ineffective |
Our strategy to extract and identify pathogenic epitopes was linked to the notion that in a systemic disease such as RA, the epitope would be present in the APCs of PBMCs as well as in synovial cells. Recently, the presence of pathogenic-like T cells has been demonstrated in the blood stream of adult forms of autoimmune arthritis and juvenile chronic arthritis; these T cells share genotypic and phenotypic characteristics with synovial T cells [
28]. Thus, the presence of pathogenic lymphocytes implies the presence of APCs as well.
The ultrafiltration of lysate and/or the suspension of cellular fragments immediately after centrifugation were intended to retain the proteolytic enzymes and allow numerous unknown small molecules, including the possible autoantigen, to pass into the ultrafiltrate aqueous solution that was used for subcutaneous injection. This extremely simplistic theory is supported by the development of an indisputable efficacy of the ultrafiltrates in patients with arthritis. Most notably, the therapeutic effect lasting 30 days from a subcutaneous dose is a type of response that differs completely from all known anti-arthritic therapies [
29].
In comparison, the administration of a specific, known autoantigen in appropriate doses, such as glutamic acid decarboxylate (GAD 65) in the non-obese diabetic rat, a well-known animal model of spontaneous autoimmune disease, has been shown to cure the disease [
14].
In the present study, the two immunotherapeutic vaccines prepared from the rheumatoid PBMCs, UF-f/t and UF-R, reached different outcomes in the two diseases; the UF-R was unable to modulate the animal model of arthritis but was effective in patients with RA while the UF-f/t worked both in AA of rats, reducing the severity of oncoming arthritis, and in patients with RA leading to an improvement of arthritic symptoms (Tables
1,
5).
The other two ultrafiltrates, UF-S and UF-sf, exhibited immunotherapeutic activities comparable to that of the ultrafiltrate prepared by freeze/thawing of PBMCs (Table
5).
It is reasonable to assume that there are at least two unknown immunoactive epitopes, in both mononuclear cells and synovial fluid ultrafiltrates.
These epitopes are thrown out of disrupted cells, specially from their endosomal compartment, during the preparation of ultrafiltrates. They are not joined to any HLA DR molecule, they pass through an ultrafilter with a cut off of 10 kDa (see “
Methods” section) and are free of molecules involved in the endocytic processing of antigens (e.g., class II-associated invariant peptide).
The Rifamycin SV might have tied the epitope effective in AA, while preparing ultrafiltrate UF-R, but leaving intact the epitope effective in human arthritis. This results in the disabling of its property to protect rats from experimental arthritis.
The epitopes (two or more) of synovial fluid, spontaneously released following the lysis of proliferating synoviocites and deeper mononuclear cell of inflamed synovium, accumulate over time. If drawn out again, just a few days after a complete extraction of the effusion, this rheumatoid synovial fluid was ineffective on prevention of AA, unlike longlasting synovial fluid (Tables
4,
5). This might explain the previous reports on depressed proliferative response by PBMCs to mHsp60 in early RA [
30]. The synovial fluids from arthrosis patients with arthrosis were always ineffective in modulating the AA of rats because they do not contain arthritogenic epitopes.
We know very little about the epitope effective in RA patients. It is present in both ultra filtrates of PBMCs and synovial fluid; it is effective as immunotherapeutic approach and exhibits immunomodulatory activities when is added to rheumatoid cell cultures (see below). We do not know the amino acid structure and the precise molecular weight. But It is just as small, much smaller than those identified with the p
an-
DR-
binding Hsp-
derived epitopes [
31], as candidate for antigen specific bystander immunotherapy [
32‐
35].
Once subcutaneously injected in RA patients, the epitope is transported by dendritic cells in inflammatory sites.
The status of the auto antigen in the ultrafiltrate, i.e., a peptidic fragment, not associated with a class II MHC molecule, might assist in explaining the early appearance of signs and symptoms of clinical improvement: being taken up and transported by dendritic cells, the self-epitope would likely interact directly with the antigen binding site of anti-idiotypic T cells on the cellular membrane (paratope), thus activating an immunoregulatory response. The idiotype-anti-idiotype network on the cell membrane is considered central in immunoregulation involving auto antigens. Therefore, the peptide would not interact with autoreactive T lymphocytes and thus would bypass the most detrimental component of the immune response in patients with arthritis. In fact, pro-inflammatory responses have never been observed in vivo or in vitro.
We hypothesized that the intrasynovial Rifamycin SV [
1,
2], through these autonomic immunological mechanisms established an endogenous immunotherapy.
The PBMCs from RA patients, in baseline cultures, were unresponsive to synthetic HSP60 bystander peptide P1 (Fig.
1) but exhibited an immune response to the addition of the ultra filtrate UF-sf which was characterized by significant increase in the expression of CTLA-4 by CD4+CD25+ (p = 0.05) and an equally significant reduction of CD69 (p = 0.04); modifications which jointly imply an immunoregulatory response that only a specific antigen is able to elicit in subjects that have, in all probability, a basic immunological trend potentially oriented toward immunoregulatory responses.
In this context, a certain role might be played by the innate immunity that is persistently activated in patients with RA [
36,
37].
The addition of autologous UF-f/t to PBMCs cultured from vaccinated patients elicited minimal change in cellular markers (Table
2). This might indicate that mononuclear cells are functionally impaired by previous in vivo stimulation. In contrast, in cultures from normal subjects there was a marked reduction in the expression of CTLA-4 (−33 %) after the addition of autologous ultrafiltrate (Table
2). This finding reinforces the speculation that subjects with RA have a particular arthritic immunological profile, particularly earliness of clinical improvement and the duration of a single subcutaneous injection.
As regards the immune response in rats with ongoing arthritis, the mechanisms of protection might be the same with the difference that, being induced, the disease is self-limiting.
However, the epitope injected with human ultra filtrate, very likely related to HSP60 molecule, remains unknown. As the HSP is an extraordinarily evolutionarily conserved molecule the presence of its epitopes in the endocytic compartment of human cells is almost obviously. It is well known that T cells specific for self-Hsp even exist in the normal T cell repertoire.
Immune response to hsp60 by rheumatoid synovial cells and the presence of these antigens in inflamed joints and subcutaneous nodules of RA patients were reported [
38,
39]. In JCA this responsiveness was directed to self hsp60 and the increased response of Hsp60-specific T-cells correlated with the spontaneous disease remission [
40].
Notably, the epitopes contained in our ultrafiltrates and responsible for the prevention of AA, appear to be smaller than the aa sequence 180–188 of the mycobacterial HSP65 which, while preventing the AA and despite having therapeutic effects on this experimental model, is not considered as the pathogenic autoantigen (it is not able to induce the disease) [
41].
The auto antigens extracted here, can be enriched many times (w/v), along with all the other small molecules, by aggregating multiple ultrafiltrates and subjecting them to lyophilization. Thus prepared, the ultrafiltrate might also be autologously used in non-rheumatological autoimmune disease, particularly in cases where the number of APCs in the blood is low, even if the autoantigen is unknown (personal obs). An adverse reaction to these antigens is unlikely given that peripheral lymphocytes reactive to self peptides are eliminated in the ontogenic selection.
In summary, this study outlines the unusual properties of the unknown and very probably pathogenic peptide that is contained in the ultrafiltrates prepared from rheumatoid PBMCs and synovial fluid. However, at the same time, the positive responses following subcutaneous administration of ultrafiltrates highlights the immunological profile of patients with RA, characterized by the potential capacity to reactivate, as a result of a specific stimulus, a pre-existing protective mechanism that is, of itself, insufficient to produce clinically significant effects.
The same human ultra filtrates contain an epitope, related to the molecule HSP60, that is able to modulate the immune mechanism implicated in AA and to stimulate protective immunity.
These studies should be considered very preliminary, and the clinical data must be interpreted with caution in view of the small number of cases.
No side effects were observed in our study following patient treatments, including at the site of subcutaneous administration of UF-sf or UF-f/t. However, on occasion the beneficial response was preceded by slight exacerbation of symptoms. Two patients in group UF-R experienced a hypersensitivity reaction after the second injection; both were characterized by skin eruptions that regressed spontaneously in a few days. In 13 patients, the intradermal sensitivity test to Rifamycin SV prior to enrollment was considered positive (appearance of wheal ≥0.5 cm). None of our patients experienced side effects common to immunotherapies in humans and in most studied experimental models of autoimmune diseases such as diabetes and multiple sclerosis (hypersensitivity reactions, immunization towards cryptic epitopes, or de novo induction of autoimmune diseases).