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Cells from a human lymphoblastoid cell line (Raji), with B-cell characteristics, and having receptors for human IgG Fc, C3b, and C3d, were used in an immunofluorescence test as in vitro detectors of immune complexes in animal and human sera. By this test, as little as 200–300 ng aggregated human gamma globulin or immune complexes per ml serum could be detected. The receptors for IgG Fc on the Raji cells were shown to be inefficient in detecting aggregated human gamma globulin and binding of aggregates to these receptors was inhibited by physiologic concentrations of 7S human IgG. Enhancement of aggregated human gamma globulin binding and binding of immune complexes formed in vitro to Raji cells was observed when the receptors for complement on these cells were used. By using the receptors for complement on Raji cells, circulating immune complexes were detected in rabbits with acute serum sickness, in mice with acute lymphocytic choriomeningitis virus infection, and in humans with immune complex type glomerulonephritis. The Raji cell test may be useful in detecting complement fixing immune complexes in different disease states, in monitoring circulating complexes in patients with immune complex diseases and in identifying the antigen(s) responsible for the induction of pathogenic immune complexes in humans and animals.

This report clearly demonstrates that a systemic lupus erythematosus (SLE)-like syndrome and lymphoma can be induced in immunologically normal (BALB/c x NZB)F1 mice by infection of neonates with a murine leukemia virus (MuLV) (Scripps leukemia virus [SLV] 60A) isolated from NZB lymphoblasts. SLV 60A was titered in vitro (XC test) and administered to newborn and adult (BALB/c x NZB)F1 mice over six log10 dilutions. Propagation of MuLV in the newborn recipients was indicated by greatly elevated serum Mu gs-1 levels which were proportional to the dose of virus given. The SLE-like syndrome was characterized by antinuclear antibodies (ANA) and immune complex-type glomerulonephritis. ANA production was related to the dose of virus and reached the highest levels at 8–16 wk. The incidence of glomerulonephritis was also correlated with the dose of virus and reached nearly 50% in the animals given the highest virus dose. Both titers of ANA and incidence of glomerulonephritis were greater in females than in males, although the amounts of Mu gs-1 in sera of both sexes were equal. The incidence of direct Coombs' positivity was not significantly affected by inoculation of this virus. The incidence and time of onset of thymocytic lymphoma were linearly related to the amount of virus inoculated. High serum Mu gs-1 levels predicted lymphoma development and reflected increases in the amount of infectious virus in the spleen. No induction of tumors, autoimmunity, or high serum Mu gs-1 levels followed administration of SLV 60A to 6-wk old (BALB/c x NZB)F1 mice or inactivated 60A or active AKR virus to newborns.

This study describes the presence of a receptor for fluid phase human C3 and C3b on Raji cell membranes. The binding of C3 and C3b was demonstrated indirectly by a fluoresceinated anti-C3 serum and directly by using radioiodinated proteins. No other complement proteins or serum factors were needed to mediate binding of C3 and C3b to the receptor. The possibility of enzymatic cleavage of C3 before or after its attachment on the cell membrane was ruled out by the demonstration of antigenically intact C3 on Raji cells. Inhibition and dissociation of Raji cell-EAC1423 rosettes by C3 and C3b indicated that both of these proteins bind to the same receptor site or closely associated receptor sites on Raji cells. C3b-bearing Raji cells were immune adherence negative, indicating that C3b binding to the receptor is brought about through the immune adherence region of the molecule and not the C3d portion. The C3 receptor on Raji cell membranes is uniformly distributed and can move on the membrane plane. Approximately 4 x 105 molecules of C3 or C3b bind per Raji cell. The receptor had a higher affinity for C3 than C3b, as was shown by uptake experiments and inhibition of Raji cell-EAC1423 rosette formation. Apart from the described receptor for C3 and C3b another specific receptor for C3b inactivator-cleaved C3b (C3d) bound to red cells was shown to be present on Raji cells. Raji cells cultured in medium containing fresh normal human serum and cobra venom factor were lysed. Similar results were obtained when C3b-bearing Raji cells were cultured in medium with fresh normal human serum. The lytic effect could be abolished by inactivating serum C3 proactivator (C3PA) and required C6. It was concluded that C3b bound to the Raji cell membrane activates the complement system through the alternate pathway and results in membrane damage and cytolysis. It is postulated that cell destruction by this mechanism may play an important role in vivo in controlling cell growth.

1. The half-lives of homologous gamma globulins labelled with I131 agree well with the half-lives of passively administered homologous antibodies and provide a simple, quantitative tool for the determination of antibody half-life when immunochemical techniques are difficult to apply. 2. The half-lives of gamma globulin or antibody vary markedly from species to species and apparently even among age groups within a species. 3. The half-lives of gamma globulin or antibody seem to be dependent, at least in part, on the metabolic rate of the host.

A secondary immune response to the soluble foreign protein antigens I*BSA and I*BGG has been demonstrated when lymph node cells, largely lymphocytes with a few reticulo-endothelial and plasma cells, from previously immunized rabbits were transferred to x-radiated recipient rabbits, and the recipients then challenged with antigen. The total specific antibody synthesized by the transferred cells during the first 8 days of the secondary response amounted to approximately ⅔ of the wet weight of the transferred cells. In an attempt to elicit a primary response, lymph node cells were obtained from normal, non-immunized donors, and transferred to x-radiated recipients. No immune response was observed upon antigenic stimulation. When normal or previously immunized lymph node cells were incubated with antigen for periods up to 1 hour, washed and injected into recipients, no antibody production was observed.

1. Repeated large infusions of heterologous plasma proteins can induce a state of specific immunologic unresponsiveness in rabbits. In normal adult rabbits this unresponsiveness in most instances lasts only about as long as the heterologous proteins are detectable in the host (3 to 4 months). In rabbits infused from the time of birth and perhaps x-radiated adult rabbits the induced immunologic unresponsiveness lasted throughout the period of observation (10 to 11 months), long after disappearance of all detectable foreign proteins. 2. This unresponsiveness appears to be specific for the antigens administered in excess and does not prevent antibody responses to even closely related antigens. 3. The unresponsiveness was not transmitted to first generation offspring. 4. The mechanism of the temporary unresponsiveness which occurs in normal adult rabbits may be dependent upon the actual presence of the antigen in the host. However, the unresponsiveness does not result from a simple neutralization of antibody, as it is formed, by the antigen, as has been suggested in the case of pneumococcal polysaccharide induced immunologic paralysis. 5. On the other hand, the mechanism of the lasting immunologic unresponsiveness developing in the "newborn" and perhaps the x-rayed rabbits may depend upon the acceptance of the foreign protein as essentially non-antigenic by the host. A similar situation is seen in the naturally occurring placental transfer of dissimilar red blood cell types between fraternal twins.

Infusions of heterologous serum proteins large enough to replace the entire normal catabolic loss of the corresponding autologous proteins in the recipient rabbits caused increased rates of plasma protein catabolism, an increase in the size of the plasma protein pool and normal or even slightly increased rates of plasma protein synthesis. The principal proteins in these infusions were catabolized at rates similar to those for corresponding homologous proteins. The most marked hyperproteinemias which developed were caused principally by increases in the host's own globulin and to a lesser extent by the presence of foreign protein in the circulation.

Daily injections of any one of several foreign serum proteins produced in rabbits functional and morphological alterations similar to those seen in acute, subacute, and chronic human glomerulonephritis. The critical factor determining whether a rabbit would develop renal disease and the type of disease developed was the amount of antibody the rabbit formed. Those responding with much antibody were likely to develop an acute, self-limited glomerulonephritis and to be subsequently immune to further renal damage. Those responding with antibody barely sufficient to neutralize the antigen injected developed subacute and chronic glomerulonephritis. In the circulation of the rabbits with chronic glomerulonephritis, there was a daily recurring antigen-antibody reaction in the region of near antigen excess to near antibody excess which presumably led to the disease. Antigen apparently in the form of antigen-antibody complexes was deposited along the renal capillary basement membranes coincident with the development of subacute and chronic glomerulonephritis. Once developed, the morphologic stigmata of chronic glomerulonephritis persisted even after injections of antigen were stopped. However, in milder instances the renal function recovered in part after stopping antigen. This experimental model has several implications: first, the renal injury is precipitated by antigens with no known affinity for, or immunologic relationship to, kidney; second, antigen antibody complexes localize in the kidney, apparently on the basis of non-immunologic factors, and may be an etiologic agent of renal injury; third, severe hypersensitivity disorders can be related specifically to relatively poor as well as to good antibody responses; and finally, the pathogenesis suggested here offers an alternative to that of nephrotoxic serum nephritis for the experimental approach to the study of human glomerulonephritis.

Tissue injury (chronic disease) associated with persistent LCM infection is apparently caused by the host immune response to the virus. Employing parabiosis or cell transfer from hyperimmune donors to isologous virus carriers, the tissue injury of chronic disease could be initiated and/or intensified. Furthermore, the transfer of anti-LCM antibody to SWR/J carrier mice results in acute necrotizing inflammatory lesions in regions of viral persistence, followed by chronic mononuclear infiltrates quite similar to those seen after the transfer of immune cells. The pathogenesis of the nonglomerular tissue injury of chronic LCM disease is apparently at least in part related to the interaction of circulating anti-LCM antibody with viral antigen at the tissue site. Trapping of circulating virus-antibody complexes in the glomerular filter is apparently the major cause of the glomerulonephritis.

Using a sheep antiserum to human glomerular basement membrane (GBM), studies of urine from healthy adults showed the presence of two cross-reactive antigens. These antigens were purified partially by preparative electrophoresis and electrofocusing, and separated on G-200; both appeared to be acidic, of high molecular weight, and carbohydrate rich. Their immunologic relationship to human GBM solubilized by several techniques was deduced from lines of identity with the native GBM digests in double diffusion analyses. These antigens will combine with homologous anti-GBM antibodies and block their fixation to human kidney sections, and will evoke heterologous anti-GBM antibody production in the rabbit. Fractionation studies of normal human serum indicated the presence of trace amounts of basement membrane antigens in the circulation. Although the serum antigens appear immunologically identical to the urinary antigens, the precise anatomic structures from which both are derived is not certain. Demonstration of immunoreactive basement membrane antigens in the circulation provides a plausible source of immunogen for the potential development of anti-GBM antibody-mediated glomerulonephritis as well as a clue to a mode for reestablishment of tolerance in such an autoimmune disorder.

The immune responsiveness of (NZB x NZW) F1 hybrid mice (NZB/W) has been compared with that of three other strains of mice, A/J, BALB/c, and CBA/J. The antigens used included sheep red blood cells (SRBC), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and human γ-globulin (HGG). It was found that important strain differences existed in the amount of antibody produced, but the relative immune responsiveness depended very much upon the nature of antigen. By comparison with the other strains tested, NZB/W mice had a higher antibody production to some antigens (SRBC and BSA) but were low responders to others (KLH). Induction of unresponsiveness to HGG by treatment with ultracentrifuged HGG was studied in the strains cited above. NZB/W mice became tolerant after injection of HGG ultracentrifuged at 100,000 g for 2 hr. Similar experiments carried out with another preparation of HGG (centrifuged at 20,000 g for 30 min) failed to reveal any abnormal behavior of NZB/W mice as compared to BALB/c or A/J mice. These results do not support the concept that NZB/W mice possess a general immune hyperreactivity or a relative inability to be made tolerant to protein antigens. However, they do not rule out the possibility that these mice have a genetically determined hyperresponsiveness to some antigens, in particular to nuclear antigens.

Aleutian disease-affected mink, which are markedly hypergammaglobulinemic, show a decreased half-life of the serum gamma globulin indicating that the hyperglobulinemia is due to increased production. No evidence that the gamma globulin was antibody to the infectious agent, to autologous or isologous tissues, or to antigens the animal was responding to prior to development of the disease was obtained. The increased gamma globulin was found to be of the 6.4S variety, and gamma globulin containing protein-protein complexes of 9S to 17S and 22S to 25S classes were observed in serums of affected mink. The findings are most consistent with the Aleutian disease virus acting as a direct and somewhat selective stimulus to plasma cell proliferation. There is no evidence that the arterial and glomerular lesions of Aleutian disease have an immunologic pathogenesis. It seems possible that these vascular changes may be directly caused by the viral agent, or may be the result of the increased gamma globulin levels.

Rats with nephrotoxic serum nephritis were studied for the presence of a possible autoimmune response to renal antigens formed and/or liberated during the immunologic reactions taking place in the glomeruli. The experiments consisted of transplantation of a normal isologous kidney to a nephritic rat and parabiosis of a normal rat to a nephritic rat. Neither functional nor morphologic abnormalities were noted in the normal kidneys in either situation. Transfer of the rabbit nephrotoxic antibody to the normal kidneys was noted in both experiments, indicating a continual dissociation and reassociation of nephrotoxic antibody with the tissue antigens of the host. Some of the nephritic rats showed a decrease in proteinuria and a slowing in the progression of the nephritis during the period in which the normal kidney was transplanted or a normal rat was united in parabiosis.

In acute nephrotoxic nephritis, polymorphonuclear leukocytes (polymorphs) accumulated in large numbers in the glomeruli in the first 12 hours. The endothelial cells were dislodged by the polymorphs which then came to lie immediately adjacent to the glomerular basement membranes. Ultrastructural changes in neither polymorphs nor basement membranes were observed. Depletion of polymorphs in both rats and rabbits prevented the development of proteinuria. This occurred when doses of nephrotoxic globulin were employed that produced proteinurias of as much as 1800 mg/kg/24 hours in intact rabbits, or enough to yield near maximal immediate proteinuria in intact rats. In addition, measurable glomerular damage was frequently averted until the onset of the secondary stage of NTN. Controls indicated that the polymorph depleted animals exhibited minimal non-specific changes in the blood, that the ability of their vascular beds to react to stimuli was not affected, and that deposition of nephrotoxic antibody and C' in the glomeruli was not inhibited. Elimination of polymorphs from the circulation was only partially effective in preventing glomerular damage when large doses of nephrotoxic globulin were used. This indicated that under these circumstances, a polymorph independent glomerular injury may also take place in first stage nephrotoxic nephritis. An indirect role of C', i.e., the accumulation of polymorphs, in bringing about glomerular injury in first stage nephrotoxic nephritis was apparent. When rabbit nephrotoxic globulin was injected into rats depleted of C', or when duck nephrotoxic globulin that fixed C' poorly was injected into normal rats, C' failed to bind with the antibody along glomerular basement membranes and polymorphs did not accumulate.

Rats with nephrotoxic serum nephritis may show fixation of complement (C') in the glomeruli during two periods. The first period occurs immediately after the injection of the antisera and extends until the host response occurs. The second period is related to the deposit of rat gamma globulin in the glomeruli presumably occurring at the time of the immune response to the heterologous nephrotoxic serum (NTS). This second period terminates about 2 to 3 weeks after injection of NTS, presumably at the time the rat stops making antibodies to the nephrotoxic gamma globulin fixed in the glomeruli. After this period, the glomerular lesion is incapable of fixing detectable amounts of circulating C' unless the antibody response to the heterologous globulin is reactivated. The immunological events which take place in the initial 2 to 3 week period appear to produce a permanent and irreparable lesion in the glomeruli leading to chronic nephritis. Rabbit NTS produces fixation of rat C' during both periods. Duck NTS shows no detectable fixation of rat C' until rat gamma globulin fixes in the glomeruli at the time of the host immune response to the heterologous nephrotoxic gamma globulin. Decomplementation of rats injected with rabbit NTS produces an amelioration of the initial period of nephrotoxic serum nephritis. The fixation of C' in the glomerular lesions was studied with the use of fluorescent antibody methods. The detection of host beta-1C globulin and gamma globulin in the glomerulus pointed to an immune reaction but not necessarily an active one. The tissue C' fixation test using kidney slices and guinea pig C' proved to be a more sensitive method of detecting tissue reactants capable of fixing C', but its relationship to in vivo events is not certain.

The role of polymorphs in the Arthus type hypersensitivity vasculitis has been studied. Polymorphs were found to play an essential role in not only producing the inflammatory vasculitis, but also were instrumental in ridding the damaged vessel of the antigen, probably by means of proteolytic catabolism at the inflammatory site. A temporal relationship between the disappearance of antigen from the damaged vessels and a decrease in inflammatory reaction was found. The earliest localization of antigen and its associated rabbit globulin in the Arthus vasculitis was found to be beneath the endothelium of small vessels. Since submitting this article for publication, Sorkin and Boyden (J. Immunol., 1959, 82, 332) have reported the catabolism of antigen in the presence of antibody by mononuclear cells obtained from the peritoneal cavities of guinea pigs. Evidence was presented indicating that the antigen molecule was actually broken down by the mononuclear cells. Techniques similar to those reported here were used.

Enhancement and acceleration of the antibody response can be achieved in some situations with large, cytotoxic doses of whole body x-radiation given after antigenic stimulation. The timing of radiation is critical and varies with the kind and physical form of the antigen. It is postulated that the x-radiation depletes the cells of the lymphoid tissues just prior to the rapid proliferation of the antigen stimulated cells. In this situation those surviving cells which are stimulated by antigen multiply more rapidly than their non-stimulated counterparts and disproportionately repopulate the depleted lymphoid tissues. Whether the x-radiation produces other effects contributing to the multiplication of the antibody forming cells was not determined. It appears that x-radiation produces its enhancement of antibody formation by means different from those operating in endotoxin and colchicine induced enhancement.

The primary phase of nephrotoxic serum nephritis produced by rabbit nephrotoxic serum appears to be dependent to a great extent, but not completely, upon the participation of serum complement. On the other hand, duck nephrotoxic serum produces its primary renal injury without detectable utilization of or dependence upon serum complement. The secondary phase of nephrotoxic serum nephritis appears to be largely or entirely dependent upon the host's antibody response to the heterologous gamma globulin fixed in the glomeruli. No evidence could be obtained for the existence of an autoimmune antikidney response by the host in this experimental model.

It has been shown that cells from neonatal rabbits are capable of producing considerable amounts of antibody after exposure to antigen in vitro and transfer to x-radiated adult recipients. This suggests that the immunological inadequacy of these neonates is not dependent primarily upon the lack of cells capable of antibody formation. Both in vitro sensitized neonatal lymphoid cells and in vivo sensitized adult lymphoid cells made much more antibody after transfer to x-rayed adult recipients than to neonatal recipients. Possible reasons for and significance of these observations are pointed out in the discussion.

X-radiation appears to exert its inhibitory effect on the antibody response by two mutually dependent routes: (a) direct radiation injury to the antibody-producing lymphoid tissue, and (b) indirect effects of altered homeostasis in the radiated host on antibody-producing tissues. Neither of these two effects alone produces significant inhibition of the secondary antibody response made by transferred lymphoid cells. However, 400 to 500 r administered in vitro to the transferred cells, plus 400 r whole body x-radiation of the recipient prior to transfer, completely inhibited the antibody response.

The immunohistochemical composition of amyloid deposits in secondary human amyloidosis and experimental amyloidosis in rabbits was studied by means of the "fluorescent antibody" technique of Coons et al. Quantitative studies of the relative amounts of gamma globulin present in the amyloid deposits by the use of radioiodinated fluorescent antibody are reported. It is concluded that amyloid deposits in several organs from cases of secondary human amyloidosis and experimental amyloidosis in rabbits contain considerable concentrations of gamma globulin. The presence of gamma globulin in amyloid might be interpreted as either a metabolic deposition of circulating globulin present in high concentrations in the plasma or as a result of an immunologic reaction involving antigen and antibody.

Lymph node cells capable of either primary or secondary antibody responses following transfer to adult normal or x-radiated homologous recipients make no response following transfer to neonatal homologous recipients. On the basis of the present observations it seems that the environment provided by the neonatal recipient is unsuitable for the immunologic activities of transferred cells in the early phases of the immune response. Neonatal recipients can, however, adequately support cells transferred during the process of active antibody formation. These findings suggest that the immunologic inadequacy of the neonatal animal is related to its internal environment and not necessarily to the lack of cells capable of antibody synthesis.

Determinations of the rates of antibody synthesis during first, second, and hyperimmune responses to bovine gamma globulin using S35-labelle aminod acids indicate the following: 1. In all three responses the rate of antibody synthesis increases while antigen is circulating and then begins to decline rapidly after elimination of detectable circulating antigen. 2. The initial rates of decline of antibody synthesis are approximately the same for all three responses. 3. There is a relatively persistent source of antibody production which appears after repeated stimulation and increases in proportion to the number of repeated stimuli.

Acute necrotizing inflammatory disease after intracerebral injection of LCM virus is largely dependent on the host immune response to the virus and is controlled, in part, by a dominant gene which is closely linked to the H-2 locus. The F1 hybrid (H-2q/k) from mating a susceptible SWR/J mouse (H-2q/q) to a resistant C3H/HeJ mouse (H-2k/k) is susceptible to LCM virus disease. When such hybrids (H-2q/k) are backcrossed to susceptible parents (H-2q/q), all F2 offspring (H-2q/q, H-2q/k) are highly susceptible. In contrast, hybrid (H-2q/k) backcross to resistant parents (H-2k/k) results in half of the F2 offspring being susceptible (H-2q/k) while the other half are resistant (H-2k/k). Similarly, in congenic H-2q/q and H-2k/k mice, H-2q/q mice are relatively susceptible to acute LCM disease, whereas H-2k/k are resistant.

Serum globulin from donor sheep made nephritic by immunization with glomerular basement membrane and subsequently nephrectomized contained specific kidney-fixing antibody and was capable of inducing an immediate, although transient, glomerulonephritis when injected into unilaterally nephrectomized lambs. This nephritis was characterized by immediate proteinuria, PMN infiltration into the glomerulus, and localization of γG- and ß1C-globulins in a linear fashion along the recipients' glomerular capillary walls. The nephritogenic property of the serum could be absorbed in vitro with isolated sheep glomerular basement membranes.

Mice infected shortly after birth with lymphocytic choriomeningitis (LCM) virus are not immunologically tolerant, although they carry the virus throughout life. These LCM carrier mice make anti-LCM antibody, which apparently complexes with viral antigen in the circulation and these complexes accumulate in the glomeruli. LCM carrier mice of different strains vary significantly as to concentration of detectable infectious virus in their tissue, amount and time of appearance of anti-LCM antibody, and development of an associated chronic disease. The chronic disease consists primarily of glomerulonephritis, focal hepatic necrosis, and disseminated lymphoid infiltrations. LCM carriers of the SWR/J strain contain high tissue concentrations of virus, considerable anti-LCM antibody detectable in the glomeruli by 3 wk to 2 months of age and develop chronic disease within the first 2–3 months of life. In contrast, C3H strain LCM carriers contain 1/1000 as much infectious virus, less detectable anti-LCM antibody, and have not, over a 24 month observation period, developed any detectable disease. B10D2 old and new carrier mice with intermediate amounts of virus develop chronic disease during the latter half of the first year of life. The pathogenesis of the glomerulonephritis of chronic LCM disease is apparently related to the formation of circulating virus-antibody complexes which are trapped in the glomerular filter. There is no evidence for direct glomerular injury by the virus nor for any autoimmune response by the host.

The development of glomerulonephritis in NZB/W mice is closely related to the formation of antinuclear, particularly anti-DNA, antibodies. The developing inflammatory glomerular lesions are characterized by the deposition of γG- and ß1C-globulins plus DNA and possibly other nuclear antigens, presumably as complexes, in a granular to lumpy pattern along the capillary walls and in the mesangia. Elution studies revealed the γG-globulin in the glomeruli to be largely γG2A-type antibody to soluble nuclear antigens. Enhancement of the antinuclear antibody response by active immunization of young NZB/W mice with DNA-methylated BSA hastens the development and increases the severity of the glomerulonephritis. Similarly, injections of soluble DNA into NZB/W mice with circulating anti-DNA antibodies but with as yet little nephritis causes rapid progression of nephritis.

Using passively administered isotope-labeled anti-KLH to suppress the antibody response of rabbits to KLH, we have attempted to estimate the amount of antigen actually involved in stimulating antibody formation. Single and paired label tracer studies of passively administered anti-KLH IgG indicated that from 0.7 to 2.9 µg were utilized or involved by the antigen in the course of a 90% suppression of the response to 2 mg KLH. Tracer studies of labeled anti-KLH F(ab')2 fragments revealed the retention of from 2 to 3 µg of these fragments in the entire rabbit during a 60% suppression of the response to 1 mg KLH. Based on previously determined ratios of mixtures of KLH and suppressive amounts of anti-KLH in adjuvant, the antibody utilization data were converted to the probable amount of antigen or immunogen involved. It appears that after an injection of 2 mg of KLH approximately 0.2–0.5 µg of antigen persisted and reacted with antibody given 24 hr later. Since all of this persisting, reactive antigen may not be immunogenic, the above estimate of immunogen is probably high, but may serve to establish upper limits for the amounts of immunogen involved in stimulating antibody formation and provide a meaningful frame of reference for antigen tracer studies.

The pathogenetic mechanism involved in a form of experimental allergic glomerulonephritis induced by immunization of rats with renal tubular antigen has been investigated. A single immunization with less than a milligram of a crude renal tubular preparation, probably containing less than 25 µg of the specific nephritogenic antigen, is effective in the induction of this form of chronic membranous glomerulonephritis. In the nephritic kidney autologous nephritogenic tubular antigen is found in the glomerular deposits along with γ-globulin and complement. When large amounts of antigen are injected during induction of the disease the exogenous immunizing antigen can also be detected in the glomerular deposits. It appears that this disease results from the formation of circulating antibodies capable of reacting with autologous renal tubular antigen(s) and the deposition of these antibodies and antigen(s) plus complement apparently as immune complexes in the glomeruli. This pathogenetic system has been termed an autologous immune complex disease and the resultant glomerulonephritis has been similarly designated.

The nephritogenic antigen, responsible for the immunogenic stimulus in experimental allergic glomerulonephritis induced with tubular antigen, has been identified as a renal tubular epithelial (RTE)-specific antigen and has been isolated in a relatively purified form. This antigen, RTE-α5, is a distinct and antigenically specific lipoprotein of high density which is derived primarily from the brush border of proximal convoluted tubular epthelium of the rat kidney. It has been suggested that this molecule may be a plasma membrane subunit. Immunization of rats with as little as 3 µg N of RTE-α5 in complete Freund's adjuvant has effectively induced this form of membranous glomerulonephritis. RTE-α5 is not a constituent of normal rat glomeruli; however, with the onset of autologous immune complex nephritis it is deposited in a granular fashion along glomerular capillary walls indistinguishable from the deposits of γ-globulin and complement. The antigenic specificity of this antigen and its tissue derivation has been explored, and the observations support the autologous immune complex pathogenesis of the glomerulonephritis induced in rats by immunization with renal tubular antigen.

These observations established the presence of anti-GBM antibodies in the sera and/or kidneys of six humans with glomerulonephritis. Further, it seems clear that these antibodies do combine with the host's glomeruli in vivo and with GBM antigen of several species in vitro. Transfer of acute glomerulonephritis to normal recipient monkeys was possible with serum or renal eluate IGG from the three patients with anti-GBM antibodies in whom sufficient material was available. Based on this transfer of nephritis and on the presence of these antibodies at the site of injury in the nephritic kidneys of both the patients and the recipient monkeys, it seems likely that they are at least a contributing, if not primary, cause of the glomerular injury. The frequency of anti-GBM antibodies in human nephritis is not certain, but on the basis of preliminary observations it would appear that they are present in all cases of Goodpasture's nephritis and somewhat less than half of the cases of subacute and chronic glomerulonephritis of adults. The nature and source of immunogen stimulating the production of anti-GBM antibodies is not known, but the presence of potentially nephritogenic GBM antigens in normal urine raises the question of possible autoimmunization. From a practical point of view, it appears that patients forming anti-GBM antibodies may not be good candidates for renal transplantation since they are likely to produce in the transplants the nephritic changes already suffered by their own kidneys.

Suppression of the primary response of rabbits to intravenously administered KLH can be achieved with very small amounts of hyperimmune anti-KLH administered a day later since the rabbit apparently rapidly eliminates most of the KLH by nonimmunologic means. The amount of passive anti-KLH needed to achieve immunosuppression was directly proportional to the dose of injected antigen. Antibody passively administered as much as 6–8 days after antigen still can be strongly immunosuppressive, which suggests that the antibody must be reacting with immunogen in or on responding cells or perhaps in the process of transfer between cells. There was no evidence that the presence of passively administered hyperimmune anti-KLH prior to the injection of antigen had any immunosuppressive action beyond the direct neutralization of the injected antigen. When KLH was injected in Freund adjuvant, anti-KLH incorporated with the KLH in the adjuvant was much more efficient in causing immunosuppression than anti-KLH given intravenously. The primary responses to 2 mg KLH given intravenously and 2 µg given in adjuvant reached approximately equal peaks and were suppressible by comparable amounts of intravenously administered anti-KLH. Two observations suggest that passive antibody neutralizes the immunogenic stimulus at the level of individual antigenic determinants and not merely by aggregating or precipitating entire antigenic molecules. First, anti-abalone hemocyanin (AH) which cross-reacts approximately 50% with KLH was only partially immunosuppressive even in extremely large amounts, i.e., amounts which could react with and precipitate much more KLH than could the smaller but more suppressive doses of anti-KLH. Second, when KLH and anti-KLH were given together in adjuvant, effective immunosuppression was achieved only with amounts of anti-KLH sufficient to saturate or cover virtually all available antigenic determinants. The immunosuppressive quality of passive antibody increases with time after immunization and with repeated immunization of the donor. In view of their relatively weak immunosuppressive properties, antibodies formed in the first weeks of a primary response may not contribute significantly to the turning off of the antibody response. In any event, results obtained by passive transfer of hyperimmune antibody to animals early in a primary response cannot be applied to the natural events in a primary response.

Rabbits immunized to several homologous renal antigens developed a variety of autoantikidney antibodies. Some of these antibodies reacted with the host's glomeruli and appeared to cause glomerulonephritis. Passive transfer of sera from some of these nephritic rabbits into normal, unilaterally nephrectomized rabbits led to the induction of nephritis. The production of autoantibody to glomeruli was transitory in most instances in spite of continued immunization. In some rabbits immunized to whole kidney homogenate, extracts or sediment, antibodies were found fixed to renal tubular basement membranes where an ultrastructural lesion was demonstrated. Rabbits also produced antikidney antibody apparently to tubular cytoplasmic components which did not fix to kidney in vivo and were of no pathogenetic significance. Immunization with autologous renal basement membranes induced a small autoantibody response in half the rabbits. This response was not associated with detectable renal injury.

Chronic viral infections of animals associated with immune complex disease resemble a number of human disorders. At present, on the basis of showing (a) virus, host antiviral antibody, and C3 deposited in a granular pattern along the glomerular capillary wall and in the mesangia and (b) virus-host Ig (presumably antiviral antibody) complexes in the circulation, immune complex disease occurs in chronic murine infections with LCM, LDV, MSV, and ADM. In addition, granular deposits in the glomeruli in Coxsachie B, polyoma, other leukemic viral infections in mice, equine anemia, and hog cholera suggest that immune complex disease also occurs in these infections. In transplacental LCM infections maternal antiviral antibody transferredin utero or via milk induces very early and severe immune complex disease. The severity of immune complex nephritis in mice neonatally or transplacentally infected with LCM virus is directly proportional to the amount of Ig elutable from the kidney and to the proportion of this Ig which reacts with the infecting virus.

Observations based on elution of IgG from nephritic kidneys of NZ mice and absorption of the eluted IgG with selected antigens indicate that their immune complex nephritis involves at least two kinds of antigen-antibody complexes. Antibodies reactive with nuclear antigens account for nearly half of the IgG eluted from the kidneys while antibodies reactive with Gross viral antigens make up a significant but lesser amount. Superimposed chronic viral infections affect the nephritis of NZ mice in different ways. LCM and polyoma infections hasten and intensify the antinuclear antibody responses and glomerulonephritis of these mice while LDV infection appears to protect against both antinuclear antibody formation and development of nephritis.

The amounts of BSA (immune complex) deposited in glomeruli of rabbits with acute and chronic serum sickness were determined using radio-labeled BSA and paired-label RSA. In animals with acute serum sickness and severe glomerulonephritis, about 18 µg of I*BSA were present in both kidneys after immune elimination (>99% of the circulating I*BSA). Visible with an immunofluorescent sensitivity of 0.25 µg per g of kidney, the I*BSA became rapidly undetectable and was presumably covered by deposits of circulating host anti-BSA and complement which increased demonstrably. In chronic serum sickness produced by daily injections of a quantity of BSA to balance antibody production, 0.04% of the daily dose of I*BSA was deposited in the glomeruli during the developmental stages of glomerulonephritis. Coincident with the onset of overt proteinuric glomerulonephritis, the daily I*BSA deposition, rate increased to about 0.5% of the daily dose. The half-disappearance rate of I*BSA from the kidney was about 5 days. Administration of huge excesses of BSA caused a fivefold increase in the half-disappearance rate. The accelerated I*BSA disappearance could be correlated with vanishing glomerular immunofluorescent deposits of BSA and immunoglobulin, as well as electron microscopic dense deposits; however, functional improvement of the disease was not observed. The roles of immune complex size, precipitating and nonprecipitating (total ABC) antibodies, and RES function in the development of chronic serum sickness glomerulonephritis are discussed.

Early, after in utero infection with LCM virus, SWR/J and HA/ICR mice developed manifestations of immune complex disease. Observations based on nursing such mice with virus-infected, immune, or noninfected mouse mothers indicated that maternal antiviral antibody was responsible for the early immune complex glomerulonephritis. Despite comparable viral persistance, in utero-infected offspring failed to develop glomerulonephritis when nursed by noninfected mouse mothers, but did when suckled by virus-infected mouse mothers. Nursing by mouse mothers carrying high titers of anti-LCM viral antibody markedly enhanced the Ig glomerular deposits and the resultant nephritis.

The half disappearance time for detectable plasma membrane-associated and cytoplasmic immunoglobulin after treatment of continuously growing diploid lymphocytes with inhibitors of protein and RNA synthesis was studied. Also, the amount of plasma membrane-associated and cytoplasmic immunoglobulin of synchronized cells in the G1 phase of the cell cycle has been studied. Plasma membrane-associated immunoglobulin has a half disappearance time of 45 min after inhibition of protein synthesis. By contrast, after treatment of cells with actinomycin D for 24 hr, plasma membrane-associated immunoglobulin remains relatively unchanged whereas cytoplasmic immunoglobulin decreased by almost 90%. In the G1 phase of the cell cycle, plasma membrane-associated immunoglobulin and cytoplasmic immunoglobulin were 70 and 10%, respectively, of that in logarithmically growing cells, and the half disappearance of M-Ig after treatment of cells with puromycin was again 45 min. In toto, these results suggest that perhaps secreted and plasma membrane-associated immunoglobulin may be separately controlled by the cells.

Chronic infections induced at birth with either LCM, an RNA virus, or polyoma, a DNA virus, in NZB, NZW, and NZB x W mice enhance ANA formation, aggravate the immune complex glomerulonephritis, and increase the associated mortality. The ANA titer was increased without apparent change in specificity of the antibodies involved in all three types of mice. Glomerulonephritis, while more severe in infected mice, was of the same type as occurred spontaneously and was characterized by a granular to lumpy accumulation of host IgG and C3 in the mesangia and along the capillary walls of the glomeruli. Of the LCM infected mice of all three types over 50% had died of glomerulonephritis by 6 months and over 85% by 9 months. Of the polyoma infected mice of all three types approximately 20% had died of glomerulonephritis by 6 months and over 40% by 9 months. Of the uninfected controls of all three types less than 10% had died by 6 months and less than 20% at 9 months except for the NZB x W females which had a 67% mortality at 9 months as a result of their spontaneous glomerulonephritis. The two viral infections had significant effect on the incidence of anti-red cell antibodies or the severity of autoimmune hemolytic anemia in any of the three NZ mice.

C3H mice chronically infected with LCM virus were found to be lethally affected by small doses of immunosuppression which caused bone marrow aplasia but had no effect on the amount of virus carried by the mouse. Humoral immune response of SWR/J mice to acute LCM infection was found to be totally suppressed by repeated single doses of 300 R/wk with no alteration in the level of virus carried by the mouse. In contrast, the established anti-LCM humoral immune response encountered in mice chronically infected with LCM virus was not suppressed by the same irradiation procedure. Over half of the chronic LCM carrier SWR/J mice treated with cyclophosphamide for 6 mo had total anti-LCM humoral immunosuppression, but showed no change in the level of virus carried. The glomerulonephritis which occurs in chronic LCM carrier mice was prevented by cyclophosphamide treatment in 90% of the mice. The humoral immune response which occurs in chronic LCM carrier mice appears to play no role in controlling the amount of virus carried by the mouse. Suppression of the LCM immune response by cyclophosphamide does prevent the development of glomerulonephritis in these mice.