The Journal of Experimental Medicine

Gabbiani, G.; Hirschel, B. J.; Ryan, G. B.; Statkov, P. R.; Majno, G.

doi: 10.1084/jem.135.4.719pmid: 4336123

Contracting granulation tissues contain fibroblasts that develop characteristics typical of smooth muscle: ( a ) They contain an extensive cytoplasmic fibrillar system. ( b ) They show immunofluorescent labeling of their cytoplasm with human anti-smooth muscle serum. ( c ) The nuclei show complicated folds and indentations, indicative of cellular contraction. ( d ) There are cell-to-cell and cell-to-stroma attachments. ( e ) It is possible to extract similar quantities of actomyosin (having the same adenosine triphosphatase activity) from granulation tissue and from pregnant rat uterus. ( f ) Strips of granulation tissue, when tested pharmacologically in vitro, behave similarly to smooth muscle. All these data support the view that, under certain conditions, fibroblasts can differentiate into a cell type structurally and functionally similar to smooth muscle and that this cell, the "myo-fibroblast," plays an important role in connective tissue contraction. Footnotes Submitted: 31 October 1971

Feldmann, Marc

doi: 10.1084/jem.135.4.735pmid: 5062922

Of many dinitrophenylated (DNP) protein conjugates tested, only DNP conjugated to polymerized flagellin (DNP-POL) (or the structurally related bacterial flagella) elicited a primary anti-DNP response in vitro. Other DNP proteins, such as DNP-monomeric flagellin (DNP-MON), were capable of inducing secondary responses in vitro. The capacity of DNP-POL to immunize spleen cell suspensions devoid of thymus-derived cells was the reason for the greater immunogenicity of DNP-POL, since even large numbers of flagellin-reactive activated thymus cells did not increase the anti-DNP response of normal spleen cells immunized with DNP-POL, whereas the thymus-dependent response to DNP-MON was markedly increased. The capacity of various batches of DNP-POL to immunize normal spleen cells in vitro varied markedly, depending on the number of DNP groups conjugated. DNP-POL with few DNP groups conjugated was immunogenic, but even at very high concentrations did not induce tolerance. In contrast, highly conjugated DNP-POL did not immunize, but readily induced tolerance. DNP-POL with intermediate degrees of conjugation were, like unconjugated polymeric flagellin, capable of inducing both immunity and tolerance. Since DNP-POL immunizes bone marrow-derived lymphocytes (B cells) directly the reduced response was not due to a masking of carrier determinants, necessary for cell collaboration. By using mixed DNP-5-(dimethylamino)-1-naphthalyl (dansyl)-POL conjugates, it was found that the inhibitory effect of a high degree of hapten conjugated was hapten specific. Depolymerization of DNP-POL to DNP-MON, which does not induce primary anti-DNP responses, was excluded by centrifugation analysis and electron microscopy. The relationship of the degree of hapten conjugation on DNP-POL to the capacity to induce tolerance and immunity in B cells has clarified the mechanism of immunological triggering of these cells. A model of the mechanism of "signal" discrimination between immunity and tolerance in B cells, based on these findings, is proposed. Footnotes Submitted: 7 September 1971

Thursh, Donald R.; Emeson, Eugene E.

doi: 10.1084/jem.135.4.754pmid: 4111774

The lymph nodes of mice actively or adoptively immunized to sheep RBC and/or chicken RBC selectively retain long-lived lymphocytes after challenge with the appropriate antigen. This retention is demonstrable within 8 hr of the time of stimulation, though it probably begins even before this, and it is essentially complete within the first 24 hr. A similar selective retention is seen in nodes regional to the injection of some nonimmunogenic substances such as turpentine, but not others such as colloidal carbon or syngeneic RBC. In animals adoptively immunized to sheep and chicken RBC simultaneously, there is a preferential accumulation of the labeled long-lived lymphocytes of donors immunized to sheep RBC in lymph nodes challenged with sheep RBC, and a preferential accumulation of lymphocytes (labeled with a different radioisotope) from donors immunized to chicken RBC in lymph nodes challenged with this antigen. This immunologically specific component is demonstrable whether the antigen is given before or after adoptive immunization, suggesting that the only labeled cells capable of specific localization in this system are those cells that normally remain in the recirculating pool. In the present experiments, 31 out of 31 sets of antigenically stimulated lymph nodes have shown radiochemical evidence of immunological specificity in the distribution of donor lymphocytes between them, while corresponding sets of nonstimulated lymph nodes have shown only small random variations in the distribution of donor cells. Two different mechanisms are postulated whereby antigenic stimulation can alter the traffic of recirculating long-lived lymphocytes through stimulated lymph nodes. One affects recirculating cells of a particular immunological specificity, while the other affects recirculating cells without regard to their immunological specificity. Footnotes Submitted: 31 October 1971

Cantor, Harvey; Asofsky, Richard

doi: 10.1084/jem.135.4.764pmid: 4401604

Two types of thymus-derived (T) lymphocytes have been shown to cooperate in the induction of graft- versus -host responses. One cell type is found in highest concentrations in the peripheral blood and lymph node, is extremely sensitive to anti-thymocyte serum (ATS) in vivo, and is probably part of the recirculating lymphoid cell pool (3). The second cell type, found in highest concentrations in the thymus and spleen, is relatively resistant to small doses of ATS in vivo. Both cell types are substantially depleted after neonatal thymectomy. Moreover, since synergism was also obtained using appropriate mixtures of cells from either parental strain in F 1 hosts, it was possible to show that the nonrecirculating cells determined the specificity of the response and were probably the precursors of effector cells in this response. The recirculating T cell appeared to amplify this response. The implications of these data are discussed. Footnotes Submitted: 26 October 1971

Mantovani, B.; Rabinovitch, M.; Nussenzweig, V.

doi: 10.1084/jem.135.4.780pmid: 5018051

Sheep red cells (E) sensitized with IgG antibody (EA) or with antibody and complement (EAC) interact in vitro with mouse peritoneal macrophage monolayers. The role of IgG and of C3 in the attachment and ingestion of the erythrocytes was examined by means of quantitative technique utilizing 51 Cr-labeled E. Controlled osmotic lysis permitted the separate measurement of the radioactivity associated with bound or with ingested E. IgG- 125 I was used to estimate the number of IgG molecules bound per E as a function of the IgG concentration. Control experiments showed that iodination did not influence the extent of binding of IgG to E and that the binding of IgG prepared from immune serum could be essentially ascribed to its anti-E antibody content. Only between 10 3 and 10 4 rabbit anti-E IgG molecules per erythrocyte were needed for detectable attachment and ingestion of EA (a maximum number of 6 x 10 5 IgG antibody molecules could be accomodated on one erythrocyte). Evidence was obtained that C3 is primarily involved in particle attachment, whereas only IgG is able to markedly promote the ingestion of particles attached to macrophages: ( a ) Addition of complement to the EA substantially increased the binding to the macrophages, whereas ingestion was increased to a smaller extent. Both binding and ingestion of EAC were markedly inhibited by papain fragments of IgG obtained from a rabbit antiserum to mouse C3. ( b ) Low doses (2 µg/ml) of papain fragments of IgG from a rabbit antiserum to mouse IgG markedly reduced the ingestion of EAC, whereas attachment of EAC to macrophages was inhibited to a much smaller degree. The possible relevance of these findings for the in vivo fate of particulate immune complexes as they interact with macrophages is discussed. It is suggested that in the primary immune response, when the complexes are predominantly in the form of EA (IgM) or EA (IgM) C3, they would tend to remain on the surface of the macrophages and thus be in a position to stimulate immunocompetent cells. In the secondary response, when EA (IgG) or EA (IgG) C3 predominate, the complexes would tend to be more rapidly interiorized and degraded by the mononuclear phagocytes, Footnotes Submitted: 7 November 1971

Biggar, W. D.; Stutman, Osias; Good, Robert A.

doi: 10.1084/jem.135.4.793pmid: 5018052

The fetal thymus at 13 days of gestation withstands transplantation and develops normally under the renal capsule of a syngenic host. Distinct differences were observed between the fetal thymus grafts and grafts from neonatal or adult thymus donors. The fetal thymus graft did not undergo the rapid and severe necrosis observed when adult thymus was grafted. Furthermore, when thymuses were transplanted into allogenic recipients, rejection was delayed. The fetal thymus was as effective as the adult thymus in restoring syngenic neonatally thymectomized mice and far superior to adult thymus when grafted into allogenic recipients. These observations seem relevant to clinical efforts to restore immunocompetence in patients with congenital absence of the thymus. Footnotes Submitted: 14 November 1971

Beer, Alan E.; Billingham, R. E.; Yang, S. L.

doi: 10.1084/jem.135.4.808pmid: 4553013

A previous report that the offspring of outbred Sprague-Dawley rats, born of mothers presensitized or tolerant with respect to tissue antigens of the Lewis strain, and reinoculated with Lewis cells during their pregnancy, reject test grafts of Lewis skin in an accelerated manner has been confirmed. This "maternally induced" alteration in reactivity of the progeny has been found to be long lasting, immunologically specific, and probably not due to transfer of humoral antibody. It has been established that the reexposure of the mothers to donor cellular antigen during pregnancy augmented the influence of the prior states of tolerance or sensitivity. To obviate the complications inherent in working with the outbred Sprague-Dawley rats, the key experiments summarized above were repeated with isogenic Fischer rats as parents and Lewis rats as the tissue donors as before. With this combination it was found that a state of prior sensitization or tolerance in the mothers resulted in the apparent induction of tolerance in some of their progeny. Reinoculation of either the tolerant or sensitized mothers during pregnancy slightly increased the incidence and degree of impairment of their offsprings' capacity to reject Lewis skin grafts. A single intraperitoneal injection of 100 x 10 6 million Lewis lymphoid cells into normal Fischer rats in the 14th–16th day of pregnancy also weakened the reactivity of their progeny to Lewis test grafts. Further to test the premise that this weakened reactivity might be due to maternal induction of tolerance, by antenatal transmission of alien cells, the lymphohematopoietic tissue system of adult Fischer females was replaced by that from Lewis donors with the aid of cyclophosphamide. It was anticipated that when these animals were mated with Fischer males, sufficient Lewis leukocytes might cross the placentas to induce high degrees of tolerance. Although normal sized healthy litters were born, about 50% of the infants succumbed to graft- versus -host (GVH) or runt disease within 40 days, many of them giving evidence of being tolerant of Lewis grafts. The mothers, too, developed chronic GVH disease. The offspring of Fischer females made chimeric with cells from (Fischer x Lewis)F 1 hybrid donors, as well as their mothers, remained healthy. Intraperitoneal injection of normal Fischer females, in the 15th–17th day of pregnancy, with 100 million lymphoid cells from specifically sensitized Lewis rats, also caused fatal runt disease to develop in about 50% of their offspring, but left the mothers unscathed. Taken together, these various findings indicate that in some genetic contexts at least the extent of the natural surreptitious transplacental cellular traffic can be considerably augmented experimentally, though how this comes about and why lymphocytic cells that are foreign to the mother can apparently gain access to fetuses more readily than her own cells remain to be determined. Footnotes Submitted: 28 November 1971

Oldstone, Michael B. A.; Dixon, Frank J.

doi: 10.1084/jem.135.4.827pmid: 4259667

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. Footnotes Submitted: 22 November 1971

Klein, George; Friberg, Sten; Harris, Henry

doi: 10.1084/jem.135.4.839pmid: 5018053

The Ehrlich ascites tumor, which has been subjected to prolonged selection for growth in allogeneic hosts, possesses powerful mechanisms for the suppression of antigens normally found on the cell surface. It has previously been shown (1, 2) that when cells in which surface antigens are fully expressed are fused with Ehrlich cells, the suppressive mechanisms of the latter continue to operate and the new surface antigens introduced into the hybrid cell by the other partner are also suppressed. In the present paper we describe the properties of hybrids in which one parent cell was the TA3 ascites carcinoma. There are two sublines of this carcinoma which originally arose as a spontaneous mammary carcinoma in a strain A mouse. The TA3/St line has a high concentration of H-2 a antigens and shows a strain-specific transplantation behavior; the TA3/Ha subline has a drastically reduced antigen concentration and readily transgresses histoincompatibility barriers. The immunoresistant TA3/Ha subline arose spontaneously without having been subjected to any known immunological selection pressure. Hybridization of TA3/Ha cells with normal diploid ACA fibroblasts reestablished full expression of H-2 a antigens in nine independently derived hybrid clones. Full reestablishment of both D- and K-end components of the H-2 a complex could be demonstrated. In some hybrid clones the concentration of H-2 a antigens was found to be comparable to that seen in (A x ACA)F 1 fibroblasts, whereas in others a higher concentration was observed, even exceeding, in some cases, the levels found in the TA3/St line. The H-2 f complex, contributed by the ACA parent cell, was fully expressed in eight of the nine hybrid clones studied. Antigen suppression thus behaves as a recessive character in the TA3/Ha hybrids, whereas in the Ehrlich hybrids antigen suppression is dominant. Footnotes Submitted: 30 November 1971

Sjöberg, Olof

doi: 10.1084/jem.135.4.850pmid: 4553014

The breaking of tolerance against the lipopolysaccharide from E. coli 055:B5 was studied. It was found that immune responsiveness recovered very slowly in vivo, tolerance still existing 3 wk after the last tolerizing injection. However, if spleen cells from tolerant mice were transferred into irradiated syngeneic recipients, the tolerant state was readily broken. Spleen cells transferred 3 days after the last tolerance-maintaining dose did not respond, whereas cells transferred on day 5 or 7 responded equally well as normal spleen cells. It was also possible to break tolerance by incubating tolerant spleen cells, which did not respond after transfer, for 20 hr in vitro before transfer into irradiated recipients. The results suggest that there exist reversibly inactivated cells in tolerant animals and that these cells can be reactivated upon removal of the cells to a neutral environment. Footnotes Submitted: 22 November 1971