Activated synovial macrophages are thought to play an important role in the pathogenesis of rheumatoid arthritis (RA) (1–3). These macrophages release proinflammatory cytokines, proteinases, and other chemical mediators that lead to the development of synovitis and joint destruction. The removal of macrophages decreases the severity of joint disease in animal models of RA (4, 5). Several antirheumatic drugs, including D-penicillamine and gold salts, affect synovial macrophages (6, 7). Furthermore, one of the actions of anti–tumor necrosis factor α (anti-TNFα) therapy may be the result of antibody-dependent cellular cytotoxicity of membranous TNFα-expressing macrophages (8, 9). Thus, reagents that target synovial macrophages may be very effective in the treatment of RA.
We have shown that folate receptor β (FRβ) messenger RNA (mRNA) and protein are highly expressed by synovial macrophages, but not by other synovial cells, in the inflamed joints of patients with RA (10). In a rat arthritis model, 99mTc-labeled folate was found to specifically accumulate in affected joints (11). Moreover, we have shown that treatment with a drug that blocks FRβ reduces disease activity in murine collagen-induced arthritis (12).
FRβ is expressed by acute myelogenous leukemia cells (13, 14). In vitro studies suggest that folate liposomes that contain cytotoxic drugs might be useful for the treatment of this disease (15). In addition, immunotoxins, which are composed of specific antibodies combined with toxins, such as Pseudomonas exotoxin A (PEA), diphtheria toxin, gelonin, saporin, and ricin A, effectively bind to malignant cells that bear the appropriate surface antigens (16, 17) and monocyte/macrophages (18–20). However, there are no reports of immunotoxins that target FRβ. Before we develop and test these immunotoxins, it is important to determine the distribution of FRβ-expressing cells in normal and inflamed tissues from humans.
In the present study, we produced monoclonal antibodies (mAb) against human FRβ. We used these mAb to detect FRβ-expressing cells in a variety of tissues and cells. We then prepared an immunotoxin composed of an anti-FRβ mAb and truncated PEA (16, 17) and verified that the immunotoxin selectively targets RA synovial macrophages in vitro.
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We produced 2 mAb that react with FRβ but not with FRα. Immunohistochemical analysis revealed FRβ-expressing cells in a wide variety of human tissues; most of the FRβ-expressing cells also expressed the macrophage marker CD163. However, FRβ was not detected on peripheral blood leukocytes, even after in vitro stimulation with LPS, M-CSF, or IFNγ. Previous studies using a polyclonal antibody and Northern blotting techniques indicated that myeloid cells and activated macrophages express FRβ antigen and mRNA; however, FRβ on the myeloid cells was not functional (14). It is intriguing that epitopes recognized by our mAb are absent on myeloid cells. This suggests that these epitopes might be associated with the function of FRβ.
It has been reported that mice with the Folbp-2 deletion, which is equivalent to FRβ, showed normal growth (30). Furthermore, folic acid has a higher affinity for FRα than for FRβ. At present, the physiologic role of FRβ on hematopoietic cells is not well defined. We recently showed that peroxynitrite generated from nitric oxide and reactive oxide induced the nitration of folic acid; one of the products, 10-nitro-folic acid, has a higher affinity for FRβ than for FRα (31). These findings, combined with those from the current study, suggest that FRβ on tissue macrophages might be required for the incorporation of folate derivatives for the synthesis of proinflammatory proteins.
We found FRβ-expressing macrophages in the sublining layer of RA synovial tissues. In a previous study by Cauli et al (32), many 27E10+ early macrophages were found in the sublining layer, whereas 25F9+ mature macrophages were more abundant in the lining layers. Thus, the majority of FRβ-expressing macrophages in RA synovial tissues seem to be acute inflammatory macrophages. Further examinations using various pathologic tissues should help to clarify the nature of FRβ-expressing macrophages.
There are several reports about folate-containing reagents that target FRβ-expressing leukemia cells and macrophages (33, 34). However, folic acid binds more avidly to FRα than to FRβ. In addition, cells have routes for folic acid uptake other than via folate receptors. Thus, reagents with higher affinity for FRβ than for FRα should be used for targeting FRβ-expressing cells. Anti-FRβ mAb has many advantages over other methods of drug delivery via FRβ in terms of specificity. In the present study, we produced an immunotoxin composed of an anti-FRβ mAb and a truncated PEA (LysPE38QQR), which lacks the cell-binding domain but retains the translocation and the adenosine diphosphate ribosylation domains (24). After internalization and proteolytic processing, domain II functions to translocate the toxin to the cytosol; the domain catalytically ADP-ribosylates elongation factor 2 in the cytosol, leading to the arrest of protein synthesis and the induction of apoptosis (16, 17).
Although most malignant cells are actively proliferating, RA synovial macrophages in the cultures we used are nondividing. We assumed that monocyte/macrophages might be resistant to PEA, as compared with dividing tumor cells. However, in the present culture system, our immunotoxin induced apoptosis and inhibited the production of TNFα by adherent RA synovial mononuclear cells. FRβ was found only on macrophages in the adherent RA synovial mononuclear cell cultures. Moreover, macrophages are the main producers of TNFα in adherent RA synovial mononuclear cell cultures (1, 2). The immunotoxin did not induce apoptosis of cultured RA synovial fibroblast-like cells. Taken together, these findings support the notion that the immunotoxin mainly damaged RA synovial macrophages. The action of the immunotoxin is not mediated through macrophages Fcγ receptors, since the immunotoxin induced more apoptosis of the FRβ gene–transfected macrophages than of the antisense gene–transfected macrophages.
The data from the present study suggest that the immunotoxin might ameliorate joint inflammation in patients with RA by inhibiting the activity of synovial macrophages. However, improvements in the immunotoxin, such as using recombinant monomeric or dimeric Fv immunotoxins, would cause less immunogenicity and more accessibility to lesions (35, 36).
The toxic side effects of immunotoxins are of at least 2 types. One type results from damage to normal cells that express the target antigen. Another type is nonspecific and is usually characterized by damage to liver cells (37). A recent study indicated that an anti-CD64 mAb conjugated with ricin A induced apoptosis of RA synovial fluid macrophages and inhibited the production of TNFα and interleukin-1β in synovial tissue explants (20). However, the CD64 antigen is expressed on monocytes and activated neutrophils in addition to tissue macrophages. Thus, immunotoxins using an anti-FRβ mAb have advantages over those using an anti-CD64 mAb because of the limited distribution of FRβ and the low levels of FRβ expression in normal tissues (38). Severe systemic toxicity might be observed with the in vivo use of immunotoxins. Thus, we would prefer to develop strategies to target activated macrophages in the joints by local injections of the immunotoxin during the first clinical trial.
We have recently observed that our immunotoxin inhibited the growth of FRβ-expressing HL-60 cells (promyelocytic leukemia cells) implanted in SCID mice (Nagayoshi R, et al: unpublished observations). In addition to reducing the activity of RA and FRβ-expressing leukemias, the immunotoxin should reduce the activity of other diseases in which macrophage activation is involved in the pathogenesis (39).