Synergy between adjuvant arthritis and collagen-induced arthritis in rats

Adjuvant arthritis (AA) in rats is susceptible to cell-mediated passive transfer. Collagen-induced arthritis (CIA) in rats is susceptible to passive transfer with antibody to type II collagen. We report here the development of strikingly severe arthritis in Lewis rats as the result of synergy between passively transferred antibody to type II collagen from rats with CIA and concanavalin A (Con A)-stimulated lymph node or spleen cells from syngeneic rats with AA. Similar synergy was seen in rats with AA given anticollagen antibody, in rats with CIA given Con A-stimulated adjuvant spleen cells, and in rats actively immunized with CII and complete Freund's adjuvant. The synergistic process caused a very severe polyarthritis, characterized by marked swelling and erythema in all the joints of the distal extremities, with histologic and radiographic evidence of early, extensive erosion of articular cartilage. Synergy was apparent if the lymphoid cells from AA rats were given up to 1 mo after a single injection of anticollagen antibody. No synergy was seen when normal rat immunoglobulin or anti-ovalbumin antibody was substituted for anticollagen antibody, when Con A-stimulated lymphoid cells from normal rats or donors with CIA were used, or when Con A-stimulated AA lymphoid cells were irradiated before transfer. Synergy between separate immune effector mechanisms may represent a general phenomenon in the pathogenesis of inflammatory joint disease.     

Autoimmunity to type II collagen an experimental model of arthritis

We have found that intradermal injection of native type II collagen extracted from human, chick or rat cartilage induces an inflammatory arthritis in approximately 40% of rats of several strains whether complete Freund's adjuvant or incomplete Freund's adjuvant is used. Type I or III collagen extracted from skin, cartilage proteoglycans and alpha1(II) chains were incapable of eliciting arthritis, as was type II collagen injected without adjuvant. The disease is a chronic proliferative synovitis, resembling adjuvant arthritis in rats and rheumatoid arthritis in humans. Native type II co-lagen modified by limited pepsin digestion still produces arthritis, suggesting that type- specific determinants residing in the helical region of the molecule are responsible for the induction of disease. Since homologous type II collagen emulsified in oil without bacterial preparations regularly causes the disease, this new animal model of arthritis represents a unique example of experimentally-inducible autoimmunity to a tissue component.     

PASSIVE TRANSFER OF ADJUVANT ARTHRITIS BY LYMPH NODE OR SPLEEN CELLS

All adjuvant-induced arthritis has been passively transferred in a highly inbred strain of rats by transfer of viable lymph node or spleen cells, but not thymus cells, to normal recipients. After an interval averaging 4.3 days recipients developed arthritis, whereas animals actively sensitized with adjuvant never developed disease before the 9th day (average 11.3 days). The transferred disease had all of the gross and pathological characteristics of primary disease, except for a lesser severity. Control studies using non-viable cells either of lymphoidal or other tissue origin were always negative. It is concluded that adjuvant arthritis is the result of an immunologic reaction which is perhaps similar to delayed hypersensitivity. The antigen in this reaction so far remains obscure.     

IMMUNOLOGIC COMPETENCE OF THORACIC DUCT CELLS : I. DELAYED HYPERSENSITIVITY

Delayed hypersensitivity was produced in donor Lewis rats by sensitization with soluble protein antigens emulsified in complete Freund's adjuvant. Cells of their thoracic duct lymph were collected for varying periods of time and transferred intravenously to isogenic Lewis recipients. With this model the following conclusions were reached: 1. Delayed hypersensitivity was transferred by thoracic duct cells. 2. The longer the drainage of the thoracic duct, the fewer cells were needed to achieve a successful transfer. With continuing drainage the proportion of small lymphocytes decreased and large cells increased. There was, therefore, a better correlation between successful transfer of delayed hypersensitivity and the number of large cells transfused than between positive skin reactions and transfer of small lymphocytes. 3. Prolonged fistula of the thoracic duct did not diminish the skin reaction of sensitized donors to specific antigen. 4. Delayed hypersensitivity was elicited in recipients 3 wk after transfer of sensitized cells. There was evidence that delayed hypersensitivity was enhanced in recipients, possibly because of prior skin testing. 5. Total body X-irradiation abolished the lesions of passively transferred delayed hypersensitivity. Recovery of positive skin tests was observed 19 to 20 days later. 6. The lesions of delayed hypersensitivity were probably mediated by cells. There was no evidence that a circulating high affinity antibody played a role in this type of immunologic reaction.     

Humoral and Cellular Sensitivity to Collagen in Type II Collagen-Induced Arthritis in Rats

We have recently described a new animal model of arthritis induced by intradermal injection of a distinct type of collagen found in cartilage (type II collagen). Since immunologic sensitivity to collagen could play a role in the pathogenesis of this type II collagen-induced arthritis in rats, the ability of purified types of native collagens to induce cellular and humoral responses was quantified by antigeninduced tritiated thymidine incorporation into lymphocytes by collagen and passive hemagglutination, respectively. Rats injected intradermally with native heterologous or homologous type II collagens in adjuvant developed type-specific cellular as well as humoral reactivity. Types I and III collagens were less immunogenic than was type II. The latter collagen induced brisk cellular and humoral responses that were equivalent whether complete Freund's adjuvant or incomplete Freund's adjuvant were employed. Both responses could be induced by native type II collagens modified by limited pepsin digestion, indicating that they are not attributable to determinants in the telopeptide regions of the molecule. Thus, these studies demonstrate the unique immunogenic as well as arthritogenic properties of the type II collagen molecule and indicate that both result from a helical conformation of its structurally distinct alpha-chains. Further, they suggest that type II collagen may, by humoral or cellular mechanisms, provoke or perpetuate inflammation in other arthritic diseases.     

Pathogenetic difference between collagen arthritis and adjuvant arthritis

Daily treatment with cyclosporin at a dose of 25 mg/kg for 14 d gave complete suppression of the development of collagen arthritis and adjuvant arthritis in Sprague-Dawley rats during an observation period of 45 d. To study whether the immunologic unresponsiveness produced by cyclosporin is antigen specific, we rechallenged the cyclosporin- protected rats with either type II collagen or complete Freund's adjuvant (CFA) after discontinuation of cyclosporin treatment. Type II collagen-immunized, cyclosporin-protected rats did not develop arthritis in response to reimmunization with type II collagen, but, they did develop arthritis in response to a subsequent injection of CFA. Similarly, CFA-injected, cyclosporin-protected rats showed a suppressed arthritogenic reaction in response to reinjection of CFA, whereas their response to a subsequent immunization with type II collagen was unaffected. On the other hand, the rats that were treated with cyclosporin without any prior antigenic challenge could develop arthritis in response to a subsequent injection of CFA or type II collagen after cessation of cyclosporin treatment. These results indicate that specific immunologic unresponsiveness can be induced by cyclosporin in the two experimental models of polyarthritis, collagen arthritis and adjuvant arthritis, and that there is no cross-reactivity between type II collagen and the mycobacterial cell wall components. The results further indicate that immunity to type II collagen plays a critical role in the pathogenesis of collagen arthritis but that its pathogenetic role in adjuvant arthritis is insignificant.     

Studies on the transfer of lymph node cells. XII. The effect of anti-rabbit-leucocyte serum on the transfer of antigen-incubated lymph node cells

In earlier studies it was shown that if rabbit lymph node cells were incubated with Shigella-trypsin filtrate and transferred to other, recipient, rabbits, agglutinins to Shigella appeared subsequently in the sera of the recipients. However, if leucocytes from the donor rabbits were injected into the recipients at a suitable interval prior to lymph node cell transfer, agglutinins to Shigella did not appear after cell transfer. In the present study, a number of experiments were done which bear on the immunologic character of this suppressive effect on the transferred cells, the "pre-injection effect." X-irradiation of the recipients before the injection of donor leucocytes led to a decrease in the extent of the pre-injection effect, i.e. to the appearance of higher agglutinin liters, in the recipient animals. It was found possible to transfer the pre-injection effect by cells of rabbit lymph nodes draining sites of injection of rabbit leucocytes. No such effect was obtained with heated aliquots of these cell suspensions or with lymph node cells from sheep-erythrocyte-injected donors. It was also possible to transfer the pre-injection effect passively by serum. Suppression of transferred lymph node cells was observed regularly after injection of serum pooled from groups of rabbits which had been injected with leucocytes pooled from the blood of 60 to 70 rabbits. The active material in anti-leucocyte serum could be precipitated in the globulin fraction of the serum and could be removed by absorption with rabbit lymph node cells. In the course of experiments on the passive transfer of the pre-injection effect by antisera of individual rabbits to leucocytes of individual donor rabbits, evidence was obtained of the existence in the sera of some normal rabbits of antibodies to some of the rabbit leucocyte antigens.     

Transfer of experimental allergic encephalomyelitis in Lewis rats using supernates of incubated sensitized lymph node cells

Supernates derived from incubated lymph node cells of Lewis rats sensitized to guinea pig spinal cord-Freund's adjuvant transfer experimental allergic encephalomyelitis (EAE) to syngeneic recipients. EAE supernatant transfer activity (EAE-STA) is not demonstrable in supernates derived from LNC of control donors not sensitized to nervous tissue. After addition of brain antigen to active supernates, EAE-STA is not longer demonstrable.     

TRANSFER OF AN AUTOIMMUNE NEPHROSIS IN THE RAT BY MEANS OF LYMPH NODE CELLS

An autoimmune nephrosis produced in rats by repeated injections of kidney extract with Freund's adjuvant has been transferred by means of lymph node cells to recipient animals rendered tolerant by neonatal injection with spleen cells from prospective donors. Transfer of the disease was manifested in the recipients by the development of proteinuria, hypoalbuminemia, hypercholesterolemia,and histological changes. The latter consisted of glomerular epithelial swelling, increase in basement membrane material and the presence of protein droplets in the glomerular and tubular epithelium. Appropriate control experiments were negative. Attempts to transfer with serum were unsuccessful. The transfer described is believed to provide evidence for an immunological mechanism for kidney and adjuvant induced nephrosis in the rat.     

IMMUNOLOGIC RESPONSE OF NEONATAL AND OLDER RABBITS TO ANTIGENS OF RABBIT LEUCOCYTES

The immunologic response of neonatal and of older rabbits to the tissue (transplantation) antigens of pooled rabbit leucocytes was studied, the test system being the suppression of formation of agglutinin to Shigella paradysenteriae by transferred lymph node cells which had been incubated with Shigella antigen. In the active induction of the suppressive effect on the transferred cells it was found that neonatal rabbits reacted as vigorously as 1 kg rabbits to the prior injection of a given number of rabbit leucocytes pooled from prospective donors of lymph node cells. The suppressive effect was dose-related, and within the range of number of leucocytes used was similar for both age groups of recipients. An attempt to detect a difference in the response during the first few days after leucocyte injection, before the full suppressive effect is reached, failed to show any difference between rabbits of the two age groups. Since it had been found possible to transfer the suppressive effect passively with sera obtained from older rabbits injected with rabbit leucocytes, attempts were made to do so with sera obtained from neonatal rabbits injected with similar numbers of pooled adult rabbit leucocytes. No consistent suppression of transferred lymph node cells was observed with sera from neonatal rabbits, even with relatively large amounts of such serum. In sera of rabbits which had been injected with rabbit leucocytes at the age of 4 to 6 weeks, suppressive antibody could be detected. When anti-rabbit-leucocyte serum obtained in adult rabbits was injected into neonatal and 1 kg recipients at a given volume per gram of animal weight the suppressive effect of the serum was of similar extent in the two groups of recipients. In the adoptive transfer of the lymph node cell suppressive effect, by cells of lymph nodes draining the sites of injection of pooled rabbit leucocytes, it was found that the popliteal lymph node cells of neonatal rabbits were as effective as those of 1 kg rabbits. Splenic cells of neonatal rabbits were also effective, when an adequate number of rabbit leucocytes had been injected intravenously. Thus, in conferring adoptive immunologic response, as in active immunization, the neonatal rabbits were as effective as the older rabbits in their response to homotransplantation antigens, in contrast to the considerable difference in concentration of the suppressive antibody in sera of neonatal and older rabbits injected with rabbit leucocytes.     

Autoimmunity to collagen in adjuvant arthritis of rats.

Arthritis can be induced in rats by intradermal injection of oil containing bacterial derivatives (adjuvant-induced arthritis) or cartilage collagen (type II collagen-induced arthritis). It was of interest, therefore, to determine whether collagen functions as an autoantigen in rats with adjuvant arthritis. Blood mononuclear cells from the majority of rats with adjuvant arthritis exhibited enhanced thymidine incorporation to homologous types I and II collagens, as well as to purified protein derivative of tuberculin. In contrast, cells from rats remaining nonarthritic after injection of adjuvant did not respond to collagen, although they did react to tuberculin. Similar results were obtained with a radiometric ear assay used to quantify intradermal delayed-type hypersensitivity in vivo. Using passive hemagglutination, autoantibodies to these collagens and their denatured alpha-chains were frequently detected in the sera of rats late in the course of adjuvant arthritis. Rats with inflammation of a hindlimb induced by turpentine did not acquire sensitivity to collagen. These data indicate that autoimmunity to collagen is a common feature of adjuvant- and collagen-induced arthritis, both of which are considered to be mediated by immunologic mechanisms.     

Serum transfer of collagen-induced arthritis in mice

Immunization of DBA/1 mice with native chick type II collagen resulted in development of polyarthritis 4-5 wk later. Sera of these mice contained high levels of anticollagen antibodies, and immunoglobulin concentrates of their sera transferred arthritis to unimmunized recipients. Histopathologically, this passively transferred arthritis resembled the early disease of immunized donors. Immunofluorescence studies demonstrated the deposition of IgG and C3 on the articular surface but not in synovial tissue of arthritic joints. Transferred, isotopically labeled anticollagen antibodies rapidly localized to the limbs and to other cartilage-containing tissues. When transfer concentrate was administered to arthritis-resistant strains, they also developed arthritis. Indeed, immunoglobulin concentrates from rats with collagen-induced arthritis transferred arthritis to naive mice. The amount of concentrate required for transfer to B10.D2 resistant mice was reduced by immunizing them with collagen 4 wk before transfer. Although susceptibility to arthritis from immunization is H-2 linked, these studies clearly demonstrate that passive transfer of arthritis depends upon injection of specific antibody and not on other host factors.     

The nature and the specificity of mononuclear cells in experimental autoimmune inflammations and the mechanisms leading to their accumulation

The nature and specificity of the mononuclear cells in passively transferred autoimmune encephalomyelitis and adrenalitis were studied. The recipients were prepared by production of a small heat lesion in the target tissue 5 days before transfer. Within 24 hr after transfer of lymph node cells from donors sensitized with the corresponding tissue antigen, a dense mononuclear cell infiltrate developed around the lesion. When lymph node cells labeled in vitro with ³H-thymidine or ³H-adenosine were transferred, a significant number of labeled lymphocytes was found in the infiltrate at 24 or 48 hr. Lymphocytes labeled with ³H-thymidine showed a greater tendency to accumulate than cells labeled with ³H-adenosine, indicating that newly formed lymphocytes were more prone to enter the reaction than older cells. Labeled lymphocytes and macrophages of recipient origin and labeled lymphocytes from donors stimulated with B. pertussis were also shown to accumulate around the heat lesion provided the reaction had been initiated by transfer of unlabeled lymphocytes from donors sensitized to the appropriate tissue-specific antigen. In recipients which were given lymph node cells from two groups of donors, sensitized either to spinal cord or to adrenal antigens, with cells from only one group of donors labeled, equal percentages of labeled cells were found around each lesion. Thus, no evidence of preferential accumulation of specifically sensitized lymphocytes was obtained. In recipients which received whole body irradiation on the day of production of the heat lesions, 5 days before transfer of lymph node cells from appropriately sensitized donors, neither monocytes nor lymphocytes accumulated around the lesion. However, if the tibial bone marrow was shielded or if bone marrow cells were given to the recipients shortly after irradiation, inflammation developed as in normal recipients. In recipients which were irradiated 24 hr after the transfer of unlabeled lymph node cells from donors sensitized to the appropriate tissue antigen and then given labeled lymph node cells from B. pertussis-stimulated donors, labeled lymphocytes were found in the reaction 24 hr later. This accumulation occurred although virtually all the lymphocytes present in the lesion at 24 hr after the first transfer were destroyed by the irradiation. The results are interpreted as follows. The autoimmune reaction is initiated by the arrival at the site of a few specifically sensitized lymphocytes, probably on a random basis. After contact with antigen, factors are produced and released which cause the influx of monocytes and of lymphocytes, in particular newly formed ones, of various specificities. There is no preferential accumulation of specifically sensitized cells. The influx of lymphocytes appears to require the presence of monocytes or macrophages in the reaction.     

STUDIES ON TUBERCULIN FEVER : IV. THE PASSIVE TRANSFER OF REACTIVITY WITH VARIOUS TISSUES OF SENSITIZED DONOR RABBITS

Utilizing techniques of passive transfer, we have investigated the factors responsible for production of fever when tuberculin is given intravenously to specifically sensitized rabbits. The ability to develop a febrile response to tuberculin could be passively transferred to normal recipients with viable mononuclear cells from peritoneal exudates, spleen, or lymph nodes of donor rabbits sensitized with BCG. Sensitivity was usually apparent 48 hr after transfer, maximal at 7 to 14 days, and rapidly declined thereafter. Granulocytes and nonviable, sonicated, mononuclear cells from similarly sensitized donors were unable to transfer this form of reactivity. Passive transfer of reactivity was also effected with plasma and serum, suggesting that the reaction of antibody with antigen contained in tuberculin is one of the initial steps by which the host cells are activated to release the endogenous pyrogen (EP) that mediates this form of hypersensitivity fever. An intravenous infusion of granulocytes, as well as of several types of mononuclear cells from sensitized donors, made most recipients responsive to the pyrogenic effect of old tuberculin (OT) given 2 hr later. Some of these passively transferred cells, such as the granulocyte and alveolar macrophage, may be activated in vivo by OT, as they are in vitro. However, in the case of splenic and lymph node cells that cannot be activated by OT to produce EP in vitro, it seems likely that an intravenous injection of OT causes these transferred, sensitized cells to liberate an intermediate substance that either directly, or in association with antigen, activates the host's normal cells to produce EP. In support of previous suggestions that leukocytes of several types, as well as phagocytic cells of the reticuloendothelial system, serve as potential sources of EP in tuberculin-induced fever, evidence was presented that OT also activates both granulocytes and mononuclear cells from sterile exudates of BCG-sensitized donors to produce EP in vitro.     

Experimental hypersensitivity pneumonitis: influence of donor sensitization

Experimental hypersensitivity pneumonitis (EHP) can be transferred to strain 2 guinea pigs by lymphoblasts from lymph node cells from sensitized guinea pigs cultured in vitro with antigen. We sought to examine the relationship between characteristics of the donor animal and development of competence to transfer EHP. We also compared cell populations that were capable and incapable of transfer using flow cytometry and fluorescein conjugated anti-Ig to determine cell size and surface IgG + (SIg +: surface immunoglobulin-positive) cells. Lymph node cells from donor animals were cultured with a soluble extract of Micropolyspora faeni (10 micrograms/ml) for 72 hours; blasts were then isolated and transferred intravenously to syngeneic recipients. Control recipients received an equal volume of medium. Four groups of donors were used: animals systemically sensitized with Freund's adjuvant and M. faeni and challenged with two, four, or eight weekly intratracheal injections of M. faeni (2-, 4-, and 8-week group); and animals sensitized with Freund's adjuvant and normal saline and challenged with two weekly intratracheal injections of normal saline (NS group). Recipients were challenged intratracheally with M. faeni 48 hours after the cell transfer and killed 4 days thereafter. Randomly selected microscopic fields of the lung (250/animal) were judged to be normal or abnormal without knowledge of treatment. All animals were maintained in high efficiency particulate accumulator-filtered air. There was a low level of pulmonary response to an intratracheal challenge of M. faeni in animals that received media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adoptive transfer of suppression of arthritis in the mouse model of collagen-induced arthritis. Evidence for a type II collagen-specific suppressor T cell.

This study details the suppressive mechanism involved in the antigen-specific suppression of collagen-induced arthritis. Intravenous injection of 500 micrograms of soluble native type II collagen 3 d before immunization with native type II collagen emulsified in complete Freund's adjuvant resulted in animals with decreased in vitro cellular and humoral immune response to native and denatured type II collagen compared with control groups. Control groups were composed of animals preinoculated with saline and type I collagen and established the antigen-specific nature of the observed suppression. Mice with reduced immune responses to type II collagen also were observed to portray little or no erythema and edema associated with collagen-induced arthritis. Adoptive transfer experiments established the requirement of T cells for the suppression of collagen-induced arthritis. Analysis of the phenotype of responding splenic cells in chronic immunotherapeutically suppressed mice in vitro revealed that responding cells were Ly1-2+ (suppressor/cytotoxic) T cells. On the other hand, the cellular phenotype of T cells responding to type II collagen in nonsuppressed collagen-induced arthritic mice was Ly1+2- (helper/inducer T cells). The data indicate that type II collagen-specific T cells are generated on intravenous inoculation of soluble native type II collagen. These cells are observed in type II collagen-immune animals, which are nonarthritic and portray reduced humoral and in vitro cellular immune response to type II collagen. This study suggests that specific suppression of immune responses to type II collagen by T-suppressor cells can be immunotherapeutic in certain forms of arthritis.     

TRANSFER OF ALLERGIC ENCEPHALOMYELITIS IN RATS BY MEANS OF LYMPH NODE CELLS

Transfer of allergic encephalomyelitis has been accomplished by injection of lymph node cells, obtained from donor rats sensitized to spinal cord, into recipient rats pretreated neonatally with normal rat spleen cells. Transfer of the disease may be achieved most frequently when the recipients are pretreated with spleen cells of the prospective lymph node cell donors. These transfers are attributed to the use of recipients which have acquired immunological tolerance to donor lymph node cells, as a result of the spleen cell pretreatment, and in which, therefore, the donor cells can survive and function longer after transfer.     

Experimental hypersensitivity pneumonitis: transfer with cultured cells

The importance of antibody and sensitized cells in hypersensitivity pneumonitis (HP) is unknown. In an attempt to create a model suitable for investigation of the mechanisms of HP, we transferred cells and serum from sensitized (Micropolyspora faeni in Freund's adjuvant) strain 2 guinea pigs to naive animals. Cells (peritoneal exudate, lymph node, or spleen) were cultured for 72 hours with either concanavalin A (Con A, 1 microgram/ml) or a soluble extract of M. faeni (10 micrograms/ml). We then injected the cells intravenously (IV) into naive guinea pigs, skin tested with purified protein derivative (PPD), challenged the animals intratracheally (IT) with M. faeni 48 hours after the cell transfer, and killed them 4 days (IT) with M. faeni 48 hours after the cell transfer, and killed them 4 days after IT challenge. We also transferred noncultured cells and antibody-containing serum from sensitized animals. Randomly selected microscopic fields of the lung (150 per animal) were judged to be normal or abnormal. All guinea pigs were maintained in high-efficiency particulate accumulator-filtered air. Compared with control animals that received media IV, there was a substantial increase (P less than 0.01) in the extent of pulmonary abnormalities in the animals receiving lymph node cells or spleen cells cultured with M. faeni, and peritoneal exudate cells cultured with Con A. Findings in recipients of peritoneal exudate cells cultured with M. faeni, or lymph node cells or spleen cells cultured with Con A did not differ from those in the control group. In contrast to cultured cells, noncultured cells and antibody-containing serum did not transfer susceptibility. PPD skin reactivity was present only in recipients of noncultured cells and not in recipients of serum or cultured cells. We conclude that experimental HP can be transferred with cultured cells from sensitized animals and that HP appears to be a cell-mediated process.     

An arthritogenic lymphokine in the rat

A type II collagen-specific arthritogenic lymphokine has been identified in the rat. Arthritogenic factor (AF) is a 65 kD protein generated in vitro by T cells from rats with collagen arthritis, and it induces an erosive, proliferative synovitis when injected into the knee joint of syngeneic naive recipients. Complement does not appear to be required. These data identify a potential T cell-mediated effector mechanism in this model, and suggest that AF may function in other inflammatory synovial diseases.     

SUPPRESSION OF ADJUVANT DISEASE IN THE RAT BY HETEROLOGOUS ANTILYMPHOCYTE GLOBULIN

The effect of antilymphocyte globulin (ALG) on adjuvant arthritis, an immunologically induced disease in the rat, was studied. ALG was prepared from the serum of rabbits immunized against rat lymphocytes. Adjuvant arthritis was induced in rats by a single intracutaneous injection of Freund's complete adjuvant; after 9 to 12 days, all control rats developed polyarthritis. Administration of antilymphocyte globulin about the time of adjuvant injection produced marked inhibition of clinical disease. Some suppression was apparent even when ALG was started after the onset of arthritis. Rats receiving ALG remained conspicuously healthy compared to controls. Urinary findings and renal histology showed no evidence of nephritis. The results of serum corticosterone determinations made it unlikely that adrenal stimulation contributed to the actions of ALG. Antilymphocyte preparations lowered peripheral lymphocyte counts and suppressed primary antibody responses to sheep erythrocytes, but had little effect on the skin reaction to PPD, even in rats protected from arthritis. All rats given ALG developed antibodies to rabbit globulin; there was no evidence that ALG inhibited the appearance of antibodies to itself, and prior hyperimmunization of rats with rabbit globulin did not interfere with the biological activity of ALG subsequently injected. Antisera produced separately against lymph node and thymus cells had identical properties with regard to agglutination of lymphocytes and thymus cells. Administered to rats, these preparations were equally potent in lowering lymphocyte counts and suppressing both adjuvant arthritis and the primary antibody response to sheep erythrocytes. It is concluded that ALG, as used in these experiments, is a potent immunosuppressive agent without obvious toxic effects.