Antibodies to IFN-gamma prevent immunologically mediated intestinal damage in murine graft-versus-host reaction.

We have tested the hypothesis that interferon-gamma (IFN-gamma) plays a role in the enteropathy of graft-versus-host reaction (GVHR) by treating host mice with a monoclonal antibody directed at this mediator. Two models of GVHR were examined. In the mild proliferative GVHR, which occurs in adult unirradiated (CBA x BALB/c)F1 mice given parental spleen cells, anti-IFN-gamma slightly inhibited the development of splenomegaly and the activation of natural killer (NK) cells in GVHR. Anti-IFN-gamma had no effect on splenomegaly or generation of anti-host cytotoxic T lymphocytes (CTL) during the more severe GVHR in adult BDF hosts, but inhibited the weight loss and mortality normally found in this GVHR. Despite these variable effects on systemic GVHR, anti-IFN-gamma treatment abolished the crypt hyperplasia and increased counts of intraepithelial lymphocytes (IEL) normally found in the jejunum of (CBA X BALB/c)F1 mice with GVHR. In parallel, anti-IFN-gamma-treated BDF1 mice with GVHR did not develop the villus atrophy and intense crypt hyperplasia found in untreated GVHR hosts. These results support the view that IFN-gamma is essential for the development of enteropathy in GVHR and we propose that this mediator may also be involved in the pathogenesis of clinical enteropathies in man.     

Role of genetic differences between mother and fetuses in the development of a graft versus host reaction induced in the mother

Development of the graft versus host reaction (GVHR) was studied in female (CBA×C57BL/6)F₁ mice during pregnancy, and after birth or the day before mating with syngeneic, semisyngeneic, and allogeneic males. The development and outcome of the GVHR in the female mice was shown to depend on genetic differences between the donors of transplanted lymphocytes and the fetuses and also on the time of induction of the GVHR. If lymphocytes from C57BL/6 mice were injected into (CBA×C57BL/6)F₁ females after parturition or on the day before mating with males of the parental CBA line, pregnancy led to enhancement of the GVHR; if lymphocytes were injected during pregnancy, an increase in resistance to the BVHR was observed. In the case of mating with males of the contralateral parental line C57BL/6 (syngeneic with respect to the lymphocyte donors) pregnancy did not affect the development of the GVHR regardless of the time when the cells were injected.Department of Microbiology and Department of Biology, Smolensk Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 3, pp. 340–342, March, 1980.     

Phenotypes of infiltrating cells in trehalose dimycolate-induced interstitial pneumonitis.

Trehalose dimycolate is a glycolipid component of the cell walls of mycobacteria, nocardia, and corynebacteria. When trehalose dimycolate is injected into certain strains of mice, they develop interstitial pneumonitis that is characterized by mononuclear cell infiltration of the alveolar walls, intra-alveolar hemorrhages, and in some animals, granuloma formation. The disorder is seldom fatal, and in approximately 4 weeks, the lungs are normal. There is strong evidence that T lymphocytes are essential for production of interstitial pneumonitis by trehalose dimycolate, but little is known about the mechanisms of lung injury in this model. The experiments described in this report were conducted to identify the roles of the various cells that accumulate in the lungs of mice with this form of interstitial pneumonitis. We found that Mac3+ macrophages were the first cells to appear in the alveolar walls. Increases in the number of L3T4+ T lymphocytes, Lyt2+ T lymphocytes, and surface-immunoglobulin-positive lymphocytes followed, but significant increases in the number of lymphoid cells were not observed until day 7, when the pulmonary lesions were well developed. Treatment of the mice with cyclophosphamide or anti-T-cell sera significantly reduced the number of lymphoid cells in the alveolar walls but did not affect the number of Mac3+ cells and did not affect development of intra-alveolar hemorrhages. Treatment with poly(I.C) significantly decreased the number of Mac3+ cells in the lungs, and these mice did not develop pulmonary hemorrhages. We conclude that although development of pulmonary lesions in trehalose dimycolate-treated mice is a T-cell-dependent process, macrophages are also essential and are more directly involved in production of the lung injury. We postulate that the lung lesions are the direct effect of macrophage-produced cytokines, such as tumor necrosis factor.     

The cellular basis of the local graft-versus-host reaction in rat kidney

Injection of parental strain rat lymphocytes under the kidney capsule of semi-allogeneic F1 recipients causes a local graft-versus-host reaction (GVHR) characterized by a heavy mononuclear cell infiltrate and renal tubular destruction. Since the cellular events involved may have relevance to allogeneic tissue damage in GVH disease and allograft rejection, a detailed analysis of the rat renal GVH reaction was performed. A purified CD4+ lymphocyte subpopulation was as effective in mediating a local GVHR as unfractionated parental lymphocytes, but neither naive CD8+ nor specifically sensitized CD8+ lymphocytes produced a detectable renal GVHR. Mononuclear cells harvested from renal GVHR lesions induced by CD4+ lymphocytes were able to lyse natural killer (NK)-sensitive targets when tested in vitro, but showed no allospecific cell-mediated cytotoxicity. Experiments using recombinant PVG rats demonstrated that the ability of the injected cells to cause a GVHR was dependent upon a disparity in MHC class II antigens and that an isolated disparity of MHC class I antigens alone was not a sufficient stimulus to provoke a response. The use of chimaeric rats demonstrated that F1 MHC alloantigens present on kidney parenchyma (but absent on bone marrow-derived cells) were not sufficient to stimulate injected parental lymphocytes, even in the presence of markedly increased amounts of MHC antigens on vascular endothelium and renal tubular cells following in vivo administration of interferon-gamma (IFN-gamma). These results suggest that the renal GVHR in the rat is mediated principally by the interaction of parental CD4+ lymphocytes recognizing and responding to class II F1 alloantigens on bone marrow-derived cells. The resulting tissue damage is most likely a result of a delayed-type hypersensitivity (DTH) phenomenon.     

Genetic differences in reactivity of lymphoid tissue during regeneration of the liver in different strains of mice

Transplantation of lymphocytes from partially hepatectomized CBA and C57BL mice, together with sheep's red cells, into lethally irradiated syngeneic mice showed that the CBA mice acquired the ability to increase the number of antibody-forming cells much sooner after the operation (4 h) than C57BL mice (17 h). Transplantation of the lymphocytes in a semisyngeneic system led, as a result of the graft versus host reaction (GVHR), to a decrease in the number of antibody-forming cells during subsequent immunization of the recipients with sheep's red cells. The GVHR was less severe in the hepatectomized mice than in the controls. These changes also appeared sooner after the operation in CBA mice than in C57BL mice.Presented by Academician of the Academy of Medical Sciences of the USSR A. P. AvtsynTranslated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 876–878, July, 1976.     

Participation of mouse T and B lymphocytes in the graft versus host reaction

Transplantation of spleen or lymph node cells from CBA mice into sublethally irradiated (CBAxC57BL/6)F₁ mice induced the development of a graft versus host reaction (GVHR). The lymphocytes lost their ability to give this reaction after treatmentin vitro with specific sera against both mouse T lymphocytes and B lymphocytes. The development of the GVHR in mice is evidently connected with cooperative interaction between T and B lymphocytes.Department of Immunology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. M. Lopukhin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 6, pp. 713–715, June, 1976.     

Immunosuppressive activity of macrophages in mice undergoing graft-versus-host reaction due to major histocompatibility complex class I plus II difference.

In a previous report, we showed that the injection of parental CD4+ T cells into major histocompatibility complex (MHC) class II disparate F1 hybrid mice induces autoimmune-like graft versus-host reaction (GVHR) resembling systemic lupus erythematosus (SLE) and the hepatic lesion of primary biliary cirrhosis (PBC). In the present study, we examined whether or not simultaneous or subsequent injection of CD8+ T cells changes the GVHR form. When parental CD8+ T cells together with CD4+ T cells were injected into MHC class I plus class II-disparate F1 mice, autoimmune phenomena did not develop and alternatively a profound immunosuppressive state was induced. Furthermore, ongoing autoimmune-like GVHR induced by CD4+ T cells was also suppressed by later injection of CD8+ T cells. In these mice, an increase of donor type CD8+ T cells and a marked decrease of host B and T cells in the spleen were observed. The spleen cells from these mice strongly inhibited the mitogenic response of normal spleen cells against lipopolysaccharide (LPS). In vitro studies demonstrated that this immunosuppression was not induced by CD8+ T cells themselves but by macrophages which produced suppressive factor(s) by LPS stimulation. These findings were discussed with reference to suppressive mechanisms of GVHR.     

Hypersensitivity pneumonitis in man. Light- and electron-microscopic studies of 18 lung biopsies.

Light- and electron-microscopic changes produced by hypersensitivity pneumonitis were analyzed in open lung biopsies taken from 18 patients with chronic forms of the disease. The main changes observed were: alveolitis (both luminal and mural), granulomas, intraalveolar buds, and interstitial fibrosis. The cells infiltrating the alveolar walls were mainly lymphocytes. Occasionally these lymphocytes presented irregularities in the contours of the nuclear membranes and resembled Sézary cells. In one patient, a few lymphocytes were found that resembled "hand-mirror" cells. Intraalveolar macrophages often had a foamy appearance. Granulomas, present in two-thirds of the patients, differed in several respects from those in sarcoidosis: they were smaller, more loosely arranged, and poorly limited; they had a higher content of lymphocytes; and they were located more frequently in alveolar tissue than in the vicinity of bronchioles and vessels. Intraalveolar buds, also present in about two thirds of the patients, were composed mainly of fibroblasts, myofibroblasts, and macrophages in a loose connective tissue that was rich in proteoglycan material. Capillaries and epithelial cells were rarely seen in buds. Alveolar buds appear to develop by a process of disruption of the epithelial lining layer, due to alveolitis, followed by intraalveolar exudation and by subsequent intraalveolar migration of connective tissue cells interacting with macrophages. Severe fibrotic and alveolar epithelial changes were observed in four patients; milder changes were frequent in most other patients. It is concluded that hypersensitivity pneumonitis usually has distinctive morphologic features; these may help to distinguish the resultant pulmonary fibrosis from that due to other causes.     

Expression of mucosa-related integrin alphaEbeta7 on alveolar T cells in interstitial lung diseases.

The expression of alphaEbeta7 integrin has been related to the selective retention of lymphocytes in mucosal tissues of gut, urogenital tract and lung. To identify potential disease-associated alphaEbeta7 expression patterns on cells accounting for lymphocytic alveolitis in interstitial lung disease (ILD), alphaE expression on CD4+ and CD8+ T cell subsets was evaluated by dual-colour flow cytometry in peripheral blood and bronchoalveolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (IPF; n = 18), hypersensitivity pneumonitis (HP; n = 20) and sarcoidosis (n = 44) in comparison with healthy controls (n = 15). In both healthy individuals and all patient groups the proportion of alphaE-bearing T cells in peripheral blood was < 2%, whereas the vast majority of alveolar CD8+ T cells consistently co-expressed alphaE. Absolute alveolar CD8+alphaE+ cell numbers/ml were up to 30-fold increased in HP patients. Proportions of alphaE-bearing CD4+ cells in BALF were significantly elevated in IPF (74.0 +/- 2.7%) and HP (70.0 +/- 2.4%) compared with normals (30.0 +/- 1.8%) (mean +/- s.e.m.; P < 0.01). In sarcoidosis, the alphaE expression on BALF CD4+ cells displayed subgroup dependency: proportions significantly lower than normal were noted in chest radiographic stage I (14.3 +/- 1.5%), but increased proportions in stages II (50.0 +/- 3.8%) and III (64.0 +/- 4.8%). Correlations between common markers of T cell activation or BALF transforming growth factor-beta (TGF-beta ) bioactivity and alphaE expression were not noted. We conclude that the vast majority of alveolar CD8+ T cells consistently express alphaEbeta7 and that distinct patterns of alphaEbeta7 expression on alveolar CD4+ lymphocytes in sarcoidosis are related to the diverse manifestations of the sarcoid inflammatory process in the lung.     

Comparison of potentiality to induce graft-versus-host reaction with small bowel, pancreas/spleen, and liver transplantation in the rat.

Although small bowel transplantation (SBT), or pancreas-spleen transplantation (PST) often lead to lethal graft-versus-host reaction (GVHR) in experimental animals, fatal GVHR is rare after clinical liver transplantation. This study describes a modified model of SBT and PST in the rat using cuff techniques applied to the renal artery and vein of the recipient. The ability of LEW (RT1(1)) or BN (RT1n) lymphocytes accompanying intestinal, splenic, or hepatic grafts to induce lethal GVHR in (LEW x BN) F1 hybrid recipients was compared. SBT and PST experiments showed that lethal GVHR always occurred in LEW-into-F1 combination, but was much less frequent in BN-into-F1 SBT. In mixed lymphocyte reaction (MLR), LEW mesenteric or splenic T cells showed significantly higher proliferative responses against BN stimulators than did BN mesenteric or splenic T cells against LEW. Adoptive cell transfer experiments using mesenteric or splenic cells also showed that LEW cells were higher responders than BN. In contrast with SBT and PST results, a lethal GVHR was not induced after liver or pancreas grafting alone in either parent-to-F1 combination. In MLR, hepatic T cells from either parent failed to elicit a proliferative response against allostimulators. These results indicate that the occurrence of lethal GVHR is dependent upon the reactivity of parental lymphocytes against allo-antigenicity of F1 hybrids and also upon the lymphoid tissue transplanted. The lack of alloreactivity of hepatic T cells accounts for the absence of lethal GVHR after liver grafting.     

Diseases caused by reactions of T lymphocytes to incompatible structures of the major histocompatibility complex. IV. Autoantibodies to nuclear antigens.

We studied the requirements for induction of ANA formation in non-irradiated F1 hybrid mice undergoing a chronic graft-versus-host reaction (GVHR) after the injection of parental-strain lymphocytes. T lymphocytes in the donor cell inoculum were both needed and sufficient for the induction of ANA formation. For optimal ANA formation, the F1 recipient mice had to differ at H-2 from the parental donor strain. ANA belonged to the IgG1, IgG2, IgM and IgA (sub)classes of immunoglobulin. IgG ANA occurred in maximal serum titres of 1 in 5,120. ANA were not donor anti-host alloantibodies. At least some ANA were true autoantibodies, i.e. of F1 origin, because they carried the Ig allotypic markers characteristic of the F1 hybrid recipients. These findings are consistent with the concept that the pathogenic mechanism underlying autoantibody formation during the GVHR is an abnormal T-B-cell co-operation. In this process, donor T cells react against foreign histocompatibility antigens of the F1 recipient and generate non-specific help for B cells, including the autoreactive B cells.     

Suppressive influence of surgical stress on the graft-versus-host reaction in mice

The influence of surgical stress on the local graft-versus-host reaction (GVHR) in F1 mice was studied. Skin incision 1 day prior to injection of parental spleen cells produced impairment of popliteal lymph node enlargement; however, this effect was not observed when GVHR was induced 3 and 5 days after operation. Strong GVHR suppressive activity of spleen cells was observed three hours after leg amputation before a decrease in thymus weight became evident. The GVHR suppressive activity declined by six hours later, but a second peak of 60% inhibition was observed after 24 h. This suppressive activity completely disappeared by treatment with anti-Thy 1.2 and complement. This shows that the GVHR is suppressed by surgical stress, and that this suppression is due to suppressor T lymphocytes.     

Usual interstitial pneumonitis is a T-cell alveolitis

Usual interstitial pneumonitis (UIP) is an idiopathic inflammatory disorder that produces scarring of the lung parenchyma. We studied open-lung biopsies of 13 patients with UIP using immunohistological staining and monoclonal antibodies. T lymphocytes (Leu 4+) accounted for 59% of cells in the alveolar septal infiltrates in UIP and OKT8+ cells accounted for the majority of T lymphocytes in most cases. OKM1+ granulocytes comprised a smaller percentage (14%) of the alveolar infiltrates. Granulocytes were most frequent within cystic airspaces and inflamed small airways. Class II HLA (Ia) antigens were expressed on lymphocytes, macrophages, endothelial cells, and alveolar type II cells in lungs with UIP. This study demonstrates that altered immunoregulatory subsets are present in the lungs of patients with UIP and suggests the possibility that activated T cells may play a role in the pathogenesis of this disorder.     

Cellular immune reactions against pancreatic islets as a consequence of graft versus host disease.

We studied autoimmune reactions against pancreatic islets occurring as a consequence of defects of the immune system rather than after pathological changes in the endocrine organ itself. Immune dysregulation was induced by transfer of parental lymphocytes into semi-allogeneic F1 recipient mice (graft versus host reaction, GVHR). Recipients were killed 1 month after the induction of the disease. Pancreatic islets were screened by light microscopy for signs of lymphocytic infiltration (insulitis). Severe insulitis was found in all mice undergoing GVHR. The cytotoxic activity of lymphocytes was apparent in electron microscopic studies and affected only B cells. Infiltrations were not seen in the exocrine part of the pancreas nor in heart, liver or kidneys at this early stage of GVHR. It is concluded that non-specific disorders of the immune system induced by GVHR may lead to specific cellular autoimmune reactions against B cells of pancreatic islets.     

Regulation of graft-versus-host-reaction by Mlsa-reactive donor T cells.

Injection of A/J splenocytes (H-2Dd, Mlsc) into unirradiated (A/J x CBA)F1 (BAF1) host mice (H-2Dd/k, Mlsd) results in an acute suppressive graft-vs.-host reaction (GVHR), characterized by immune dysfunction and appreciable donor cell engraftment; injection of the CBA/J parent (H-2Dk, Mlsd), which recognizes no Mls disparity in the host, results in little or no GVHR. Furthermore, the Mlsa-reactive V beta 6 and V beta 8.1 T cell subsets in A/J T cells expand significantly in the GVHR host. Finally, depletion of V beta 6+ and V beta 8.1+ from the A/J population abrogates the proliferative response to BAF1 in vitro and the development of GVHR in vivo. Thus, the response to Mls determinants can contribute to the generation of a GVHR.     

Murine hypersensitivity pneumonitis: a study of cellular infiltrates and cytokine production and its modulation by cyclosporin A.

The progression of hypersensitivity pneumonitis (HP) was evaluated in mice repeatedly challenged with the actinomycete Faeni rectivirgula (Micropolyspora faeni) (90 or 180 micrograms), at the cellular level and at the mediator level. Instillation of F. rectivirgula by the intranasal route determined a granulomatous inflammation in the lungs of animals correlated with a dramatic increase (5- to 6-fold) in cellularity in the bronchoalveolar space and an increase in the percentage of lymphocytes. Disease in mice was also correlated with high spontaneous release of the cytokines interleukin-1 (IL-1) (60 U/ml), interleukin-6 (IL-6) (72 U/ml), and tumor necrosis factor-alpha (TNF-alpha) (56 U/ml) in the bronchoalveolar lavage (BAL) fluid, as well as by an enhanced capacity for cytokine release by macrophages upon stimulation with F. rectivirgula. It was also found that the pulmonary inflammation was correlated with a 60 to 70% increase in total lung weight after 4 wk and a significant lung fibrosis as seen by a 2-fold increase in lung hydroxyproline levels. Treatment of challenged mice with cyclosporin A (CyA) led to an abrogation of the disease as seen by an abrogation of the increase in lung index, lack of IL-1 and TNF-alpha release in the BAL. CyA did not, however, completely prevent the alveolitis as seen by the cellular infiltrate (2- to 3-fold in BAL cell increase). It also did not prevent the T-lymphocyte recruitment associated with HP, although these cells did not proliferate in response to the F. rectivirgula antigen, in contrast to BAL cells from F. rectivirgula-challenged mice treated with excipient only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Graft-Versus-Host Reactions in F1 Hybrid Mice: MHC-Restriction-Independent Generalized Depression of Virus-Specific Cytotoxic T Cell Response

A graft-versus-host reaction (GVHR) was induced in F₁ hybrid mice by intravenous injection of parental spleen cells. A possible effect generated by recipient F₁ mice of a GVHR on the restriction specificities of anti-viral cytotoxic T cells was investigated. (A/J × C57BL/6) F₁ recipients were injected with 1 × 10⁸ or 2 × 10⁷ spleen cells from either parent. Zero to two weeks later during an acute GVHR or 12 to 26 weeks later during a chronic GVHR, spleen cells from these F1 recipients were assayed for their capacity to generate vaccinia (or lymphocytic choriomeningitis) virus-specific cytotoxic T cells. No effect was seen when parental cells were injected with virus on the same day. During the acute phase of the GVHR, recipients of spleen cells of both doses or from either parent injected 7 or 14 days previously generated markedly fewer cytotoxic T cells with respect to both parental restriction specificities. In the more chronic situation only, recipients of 1 × 10⁸ parental spleen cells showed depressed virus-specific cytotoxic responses, again both restriction specificities were affected comparably.Therefore, a GVHR depresses generation of virus-specific cytotoxic T cells in a dose- andtime-dependent way, but does not measurably disturb the balance of parental 1 versus parental 2 restriction specificity in an F₁ recipient.     

Cyclosporin alters the induction of allospecific tolerance in vivo

CBA mice were made hyporesponsive to A/J alloantigens by either neonatal inoculation of (CBA X A)F1 hybrid lymphoid cells or by intravenous injection of adult mice with A/J bone marrow cells. Specific alloreactivity was assessed in vitro by induction of anti-A/J cytotoxic T lymphocytes (CTL) or in vivo by skin graft rejection. Cyclosporin A given at the same time as the tolerance-inducing regimen of F1 (or parental) lymphoid cells abolished the hyporesponsiveness normally induced by these injections.     

Suppression of the local graft-vs.-host reaction in rats by treatment with a monoclonal antibody specific for the interleukin 2 receptor

Local graft-vs.-host reaction (GVHR) was induced in rats by injecting parental cells into young F1 recipients. As a consequence of antigenic stimulation in the course of developing GVHR in the responding lymph nodes, the number of interleukin 2-receptor (IL 2R)-bearing T cells increased from less than 1% up to 10% of the total population. The IL 2R-bearing cells were located mainly in the T cell areas of the reactive lymph nodes. As assessed by the determination of the GVHR indices, treatment of the recipients with anti-T-helper subset-specific mAb (W3/25) or with anti-IL 2R mAb (ART-18) inhibited the GVHR. In parallel, the number of IL 2R-bearing cells was reduced to the normal levels. W3/25 mAb treatment changed the helper/suppressor subset ratio and reduced the number of circulating lymphocytes in the peripheral blood. In contrast, ART-18 mAb treatment did not induce any detectable changes in the subset distribution and it did not affect the number of circulating lymphocytes. The results demonstrate the key role that the IL 2R-positive cells play in the proliferative phase of acute GVHR, and favor the use of anti-IL 2R mAb as selective immunosuppressive agents.     

Participation of phytohemagglutinin-transformed mouse lymphocytes in the graft versus host reaction

Transformed lymphocytes obtained by stimulating lymph node cells of CBA mice with phytohemagglutinin (PHA) do not give the graft versus host reaction (GVHR) if injected into sublethally irradiated (CBA×C57BL/6) F₁ hybrids. In a population of PHA-stimulated cells the GVHR was induced by small lymphocytes having the same concentration of antigens, detectable by antilymphocytic serum, as intact lymphocytes.Department of Immunology, Medico-Biological Faculty, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. M. Lopukhin.) Translated from Byulletin' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 9, pp. 1096–1098, September, 1976.