The Stimulus to Host Cell Proliferation in Graft-versus-Host Reactions

Two experiments are described concerned with the mechanism of host cell activation in the rat popliteal lymph node (LN) undergoing a graft-versus-host (GVH) reaction. (1) Irradiated, F₁ hybrid hosts (750 rad) mounted an impaired response to parental strain T cells. This was augmented by an intravenous injection of F₁ hybrid lymphocytes but not by parental strain B lymphocytes syngeneic with the initiating T cells. When the donor T cells (footpad) und B lymphocytes (intravenous) were completely allogeneic the residual response of the irradiated F₁ was completely inhibited. (2) The popliteal LN response in the semi-allogeneic situation of the type (A × C)F₁→(B × C)F₁ was, if anything, weaker than in the allogeneic situation AA→BB. These results and other data are discussed in terms of a possible major histocompatibility complex (MHC) requirement for host cell activation. The sharing of an MHC haplotype between donor and host cells is unlikely to be a necessary or sufficient condition for host cell activation.     

Anti-Parental Lymphocyte Reactions in Neonatal Fl Hybrid Rats

The subpopulation of parental-strain lymphocytes responsible for the recognition of a particular F1 hybrid strain as foreign has been shown to be subject to specific, reversible inactivation after its injection into neonatal rats of that F1 hybrid strain. Neonates born to mothers that were syngeneic with the parental-strain lymphocytes under test acquired the capacity to inactivate these lymphocytes at an earlier age than did the genotypically identical reciprocal F1 hybrids. Neonates had little capacity to inactivate completely allogeneic lymphocytes. It is inferred from the difference in behavior between reciprocal F1 hybrids that the augmented ability to inactivate anti-F1 hybrid maternal-strain lymphocytes follows exposure to such cells in utero and to antibodies with anti-F1 hybrid activity in colostrum. Specific inactivation of those marauding maternal lymphocytes with anti-fetal activity is envisaged as an important means of protection of the fetus from immunological attack by the mother. On the basis of the results presented in this and the preceding paper, it has been proposed that many of the sequelae of the transfer of immunocompetent parental-strain cells to F1 hybrid animals result not from graft anti-host activity but from an F1 hybrid anti-parental lymphocyte response that has eluded normal regulatory mechanisms. These experiments also raise the possibility that regulation of auto-immune responses may be achieved by the inactivation of lymphocytes with anti-self reactivity by other lymphocytes that respond to the recognition structure required for such reactivity.     

Anti-Self Receptors

Murine thymocytes and peripheral lymphocytes bind autologous erythrocytes via H-2L -region-restricted receptors. After inhibiting autorosetting with different erythrocyte sonicates the specificity of these anti-self receptors was examined in F₁ hybrid and chimaeric mice. Most F₁ lymphocytes simultaneously expressed receptors against both parental haplotypes. Furthermore, analysis of lymphocytes from allogeneic and semi-allogeneic chimaeras clearly demonstrated that radio-resistant elements in the recipient thymus did not modify the haplotype specificity of the receptors on donor-derived lymphocytes.     

Proliferation of C3HXCBA Hybrid Lymphocytes in the Spleens of Irradiated CBA Mice: Linkage to the Mls-Gene

Lymphocytes from C3HXCBA F₁ mice, lacking delectable Mls-antigens, can specifically inactivate T-cell clones from the H-2-idcmical strain CBA, which are reactive against the C3H-Determined Mls-antigen. Such F₁ lymphocytes also proliferate in the spleens of irradiated CBA mice. The age dependence of the mixed lymphocyte culture stimulatory capacity and the capacity to proliferate in irradiated CBA recipients of lymphocytes from C3HXCBA F₁ mice were compared. It was observed that appearance of Mls-bearing cells is preceded by the appearance of F₁ cells with strong proliferate capacity in irradiated CBA recipients. The in vivo proliferative response is also dependent on the age of the recipient. The varying response is not reflected in the quantity ofanli-C3H Mls-reactive cells in CBA mice. A strong linkage between the genes controlling the above proliferative and stimulatory capacities of C3HXCBA lymphocytes was found.     

Phenotypical Characterization of Cells in the Thoracic Duct of Patients with and without Systemic Inflammatory Response Syndrome and Multiple Organ Failure

The subset composition and recirculation properties of the migrating lymphocyte pool in humans is largely unknown. The present study was conducted in order to phenotypically characterize cells in human thoracic duct lymph of patients under non-inflammatory and inflammatory conditions. These data were compared with data from peripheral blood, with special emphasis on those cells homing to the gut. Thoracic duct lymph and peripheral blood contained comparable proportions of B and T lymphocytes and CD8⁺ cells. Thoracic duct lymph contained proportionally more CD4⁺ cells, more CD4⁺CD45RO⁺ that express α₄β₇ cells and more CD8⁺CD45RO⁺ that express α₄β₇, as compared to peripheral blood. These data suggest an equal recirculation rate of B and T lymphocytes; a more active recirculation of CD4⁺ cells compared to CD8⁺ cells; and a more active recirculation of memory cells to the gut as compared to other extra-lymphoid sites in patients under non-inflammatory conditions. Data were also obtained in patients with the system inflammatory response syndrome and multiple organ failure. Although it is generally assumed that granulocytes and monocytes do not recirculate, lymph of multiple organ failure patients contained significantly more granulocytes than monocytes, indicating that in severe generalized inflammatory states these cells re-enter the circulation through the thoracic duct. Furthermore, no increased activation of cells homing to the gut was found in these patients.     

Differential Role of Protein Kinase C in Cytokine Induced Lymphocyte-Endothelium Interaction In Vitro

The subset composition of the migrating lymphocyte pool is largely unknown. In order to determine the number of B, T, CD8 ⁺, CD4⁺ and CD4⁺'naive'(CD45RC⁺) and 'memory' (CD45RC⁻) lymphocytes in this pool, the thoracic duct lymph of the rat was drained for 7 days. The effect of lymphocyte deplction on the number of blood lymphocytes was also monitored. In addition, the influence of continuously applied interferon-γ (IFN-α) on the mobilization of the migrating lymphocyte pool was investigated.
Within 1 week 2 × 10⁹ thoracic duct lymphocytes (TDL) were collected, which represents about 50% of the total lymphocyte pool of an adult rat. Among the migrating lymphocytes an early and a late mobilized population could be differentiated. In the former the CD4⁺'naive' (CD45RC⁺) T lymphocytes constituted the largest population, whereas in the latter it was the B lymphocytes.
Continuous infusion of IFN-γ did not affect the number of lymphocytes in the blood. In contrast, in the thoracic duct IFN-γ reduced the appearance of all lymphocyte subsets. However, the pattern of reduction over time differed markedly depending on the population (early or late mobilized) and the phenotype (B- or T-tymphocyte subsets.     

A Study on Graft-versus-Host Reaction (GVHR) by Simonsen's Splenomegaly Assay Cells and Antigen Systems Involved in Induction of GVHR

Cells and histocompatibility antigen systems involved in graft-versus-host reactions (GVHR) were analyzed using Simonsen's splenomegaly assay employing various combinations of donor and F₁ hybrid recipients mice. Most of the cells proliferating in spleens of mice undergoing GVHR were J11d⁺, and had histological features of cells of the hematopoietic lineage. The proportions of CD3⁺ T cells were decreased in the spleens. Disparity at minor histocompatibility determinants of AKR, I-E and H-2D regions between B10.A(4R) donors and (4R × AKR) F₁ recipients evoked only negligible GVHR. On the contrary, disparity at H-2K and/or I-A regions appeared to be sufficient to permit induction of full GVHR. When surface markers of donor spleen cells were analyzed, it was shown that Thy-1⁺ and/or MEL-14⁺ cells caused a strong effect on GVHR. Further, either CD4⁺ or CD8⁺ T cell subset could induce significant GVHR. However, synergistic influences of these two T cell subsets on one another in GVHR were observed. The present results raise the possibility of using Simonsen's assay along with a number of reagents to identify the contribution of subsets of T lymphocytes and in analyzing precise contributions of cellular components from both donor and recipient, and also of the target antigen systems of the recipient that contribute to early events involved in GVHR.     

The Neuropeptide Substance P does not Influence the Migration of B, T, CD8+ and CD4+ ('Naive' and 'Memory') Lymphocytes from Blood to Lymph in the Normal Rat

Thoracic duct lymphocytes (TDL) continuously patrol through the body, facilitating immune responses at most sites. The neuropeptide Substance P might regulate immune responses by influencing the migration of TDL. Therefore, it was investigated whether Substance P affects the migration of thoracic duct B, T, CDS⁺ and CD4⁺ ('naive' and 'memory') lymphocytes from blood to lymph in vivo . Labelled TDL were either incubated with Substance P and then injected into normal rats, or incubated without Substance P and then injected into rats continuously receiving Substance P intravenously. The numbers of labeled B, T, CDS⁺ and CD4⁺ ('naive' and 'memory') lymphocytes were determined in blood and thoracic duct lymph for 1 and 5 days, respectively. Neither the in vitro incubation with Substance P nor its in vivo application influenced the disappearance of any lymphocyte subset from the blood or its reappearance in the lymph. In addition, continuous intravenous application of the Substance P antagonist CP 96.345 did not alter the volume or the lymphocyte number of the efferent lymph. The present study indicates that the nervous system does not influence immune responses via Substance P by altering the migration pattern of B, T, CD8⁺ and CD4⁺ ('naive' and 'memory') lymphocytes.     

Decreased Syngeneic Mixed Lymphocyte Reaction in Autoimmune Susceptible Mice

The syngeneic mixed lymphocyte reaction (SMLR) is a proliferative response mediated by murine splenic thymus-derived (T) lymphocytes after stimulation by syngeneic non-T cells. Several murine strains genetically susceptible to autoimmune diseases have decreased SMLR compared with normal strains. This diminished SMLR occurs before the onset of clinical disease and is most pronounced in those autoimmune mice that express a severe form of the disease. For example, in NZB/NZW F₁ hybrid (B/W) mice, the SMLR is lower at 7 than at 2 months of age. The SMLR is lower in BXSB males, which develop more accelerated autoimmune disease, than in BXSB females. The defect in these autoimmune strains resides within the responding Lyt 1⁺, 2, 3⁻ T-cell population. These results suggest the presence of a common T-cell defect in several murine strains that spontaneously develop autoimmunity.     

VARIOUS EPITHELIAL AND NON-EPITHELIAL TUMORS SPONTANEOUSLY OCCURRING IN LONG-LIVED MICE OF A/St, CBA, C57BL/6 AND THEIR HYBRID MICE

Various kinds of epithelial and non-epithelial tumors, and tumor-like lesions spontaeously developed in long-lived mice of strains A/St, C57BL/6 and CBA, and of (C57BL/6 x CBA) F₁, hybrids, all of which had been fed in the 2nd Department of Pathology, Gifu University School of Medicine.
Some tumors were successfully transplanted into the same strain of mice. An interesting tumor line showing phagocytic activity and growing only in the liver even when inoculated subcutaneously, which was obtained from a reticulum cell neoplasm, Type A by Dunn, originated in the liver of a (C57BL/6 X CBA) F₁ hybrid male mouse.     

Enhanced T Lymphocyte Expression of LFA-1, ICAM-1, and the TNF Receptor Family Member OX40 in HgCl2-Induced Systemic Autoimmunity

Injection of mercuric chloride into Brown Norway (BN) rats induces a T lymphocyte-dependent autoimmune syndrome. In order to investigate whether modification of adhesion and costimulatory molecules on T lymphocytes may be involved in early T lymphocyte activation by HgCl₂, the authors analysed expression of these molecules in peripheral lymph node cells from BN rats at day 4 after injection of HgCl₂. Tri-colour flow cytometry was performed for expression analysis within CD45RC-defined subsets of CD4⁺ and CD8⁺ cells. Compared to control rats, HgCl₂-exposed rats showed increased numbers of lymphocytes, especially of T lymphocyte blast cells. The levels of LFA-1 expression as well as the fractions of ICAM-1⁺ cells were significantly increased in all CD45RC-defined subsets of CD4⁺ and CD8⁺ cells. Within the CD4⁺CD45RC^lo T lymphocyte population, HgCl₂-injected rats showed a highly significant increase in the number of cells expressing OX40, which is a member of the TNF receptor family. Moreover, only CD4⁺CD45RC^lo blast cells of HgCl₂-exposed rats showed decreased expression of CD43, increased expression of CD49d and decreased numbers of CD26⁺ cells. The results indicate that induction of autoimmunity by HgCl₂ in BN rats is associated with altered expression of T lymphocyte costimulatory molecules, predominantly on CD4⁺CD45RC^lo cells, which may be caused by a direct effect of HgCl₂ on these cells, and may precipitate further activation of T and B lymphocytes by HgCl₂     

Weak Graft-Versus-Host Response of CBA Lymphocytes Against the H-2-Identical Strain C3H

Lymphocytes from CBA and C3H mice were tested for graft-versus-host (GVH) reactivity in C3H × CBA recipients. Three different GVH assays were used, namely, the spleen enlargement test, the popliteal lymph node assay, and inhibition of allogeneic bone marrow proliferation CBA lymphocytes showed only weak reactivity against C3H X CBA tissues in all these tests, while there was strong reactivity in H-2-different F₁ hybrids. The capacity of C3H and CBA cells in producing a host-versus-graft (HVG) reaction was also tested. It was observed that CBA cells injected into the foot pads of C₃H mice induced an enlargement of the local popliteal lymph node that was as great as when C3H cells were injected into CBA mice. On the other hand, cells from the H-2-different strain 57B1 induced a considerably smaller HVG reaition when injected into CBA mice. It is concluded that antigens that strongly stimulate mixed lymphocyte cultures do not always stimulate allogeneic lymphocytes to strong GVH reactions in vivo.     

Degradation of Serum Amyloid A in Amyloid-Susceptible and Amyloid-Resistant Mouse Strains

Degradation of serum amyloid A (apoSAA) by resident peritoneal cells (RPCS) and conditioned medium (CDM), prepared with RPCS, from amyloid-susceptible CBA/J mice, amyloid-resistant CE/J mice and their amyloid-resistant CBA/J × CE/J F₁ progeny was investigated in vitro . Serum amyloid A was derived from murine acute phase (AP) plasma and associated with high density lipoprotein (HDL). Degradation of apoSAA by RPCS and CDM from CBA/J mice was complete while degradation by RPCS and CDM from CE/J mice did not occur. Degradation of apoSAA by RPCS and CDM from CBA/J × CE/J F₁ hybrid mice was indistinguishable from that by RPCS and CDM from the CBA/J parent. Intermediate fragments were not detected with either RPCS or CDM from CBA/J mice or CBA/J × CE/J F₁ hybrid mice. Degradation of apoSAA was inhibited by phenylmethanylsulfonyl fluoride (PMSF) indicating that the enzyme, secreted into the fluid phase, was a serine esterase. Unlike apoSAA, HDL-associated apoA-1 remained intact. It was thus concluded that while selective degradation of HDL-associated apoSAA (apoSAA-HDL) by RPCS from the CBA/J and CE/J mice was significantly different, the genetic study did not support the hypothesis that there was direct linkage between impaired degradation of apoSAA-HDL in the CE/J mouse strain and protection against amyloid fibril formation. As amyloid resistance in CBA/J × CE/J F₁ hybrid mice is not attributable to failure to express the amyloidogenic isoform apoSAA₂, the study supports the original hypothesis that amyloid resistance may be linked to expression of apoSAA_cej     

F1-Hybrid Anti-Parental-Strain Reactivity

Experiments were conducted 10 explore whether C3H×CBA lymphocytes can react by proliferation when they meet parental CBA spleen cells. MLC tests could not detect such a reaction, but infusion of C3H×CBA lymph node cells into irradiated CBA hosts resulted in rapid cell proliferation in the host's spleen. Such a cell proliferation was also observed after Infusion of T-cell-enriched lymph node cell preparations or thymic cells from C3H×CBA donors. Upon transfer to new irradiated mice these proliferating cells continued to proliferate in CBA mice but not in C3H×CBA mice. Further evidence that the injected T-cells were the proliferating emerged from experiments where AKR×CBA lymphocytes were found to proliferate in the spleens of irradiated CBA mice and that most of these cells possessed the these antigen determined by AKR. Proliferation of F₁-hybrid lymphocytes in the spleens of its irradiated parental strains was found not to be a general phenomenon and is probably restricted to some Mls-antigen-compatible strain combinations. The possibility that the C3H×CBA hybrid lymphocytes are stimulated to proliferation by CBA lymphocytes reactive against the C3H-determined Mls antigen is discussed.     

Role of tachykinin NK1 and NK2 receptors in allergen-induced early and late asthmatic reactions, airway hyperresponsiveness, and airway inflammation in conscious, unrestrained guinea pigs

Using a guinea pig model of allergic asthma, we investigated the effects of the inhaled, highly selective nonpeptide tachykinin NK₁ and NK₂ receptor antagonists SR 140333 and SR 48968, respectively, on allergen-induced early (EAR) and late (LAR) asthmatic reactions, airway hyperreactivity (AHR) after these reactions, and infiltration of inflammatory cells in the airways. Both SR 140333 (100 nM, 3 min) and SR 48968 (100 nM, 3 min) had no effect on the severity of the EAR, while the NK₂ receptor antagonist SR 48968, but not the NK₁ receptor antagonist SR 140333, caused significant inhibition of the LAR. SR 140333 significantly reduced the allergen-induced AHR to histamine, both after the EAR and the LAR. By contrast, SR 48968 did not affect the AHR after the EAR, but significantly attenuated the AHR after the LAR. Bronchoalveolar lavage studies performed after the LAR indicated that SR 140333 caused significant inhibition of allergen-induced infiltration of eosinophils, neutrophils and lymphocytes, while SR 48968 attenuated the infiltration of neutrophils and lymphocytes, but not of eosinophils. Both NK receptor antagonists tended to reduce the accumulation of ciliated epithelial cells in the airways. These results indicate that NK₁ and NK₂ receptors are importantly, but differentially, involved in the development of allergen-induced airways obstruction, AHR and infiltration of inflammatory cells in the airways. Therefore, both NK₁ and NK₂ receptor antagonists, or dual NK₁ and NK₂ antagonists, could be useful in the treatment of allergic asthma.     

Cell-mediated immunity to chemically xenogenized tumors. V. Failure of novel antigens to increase the frequency of tumor-specific cytotoxic T cells

Xenogenized variant cells (L5178Y/DTIC) of a murine lymphoma line confer a high degree of specific protection against subsequent challenge of mice with parental L5178Y cells. In an attempt to better define the effect of DTIC-induced determinants on parental antigen recognition and the mechanisms involved in this protection, we evaluated the frequency of anti-parental tumor cytotoxic T lymphocyte precursors following priming of mice with xenogenized cells in vivo. In addition, we tested the effect of host sensitization with the immunogenic, retrovirus-related proteins that are precipitated from the surface of L5178Y/DTIC cells by means of specific antibody. Our results indicated that the novel determinants induced by DTIC treatment on L5178Y cells do not act as helper determinants for the generation of tumor-specific cytotoxic responses. Therefore, increased frequency of tumor-specific cytotoxic lymphocytes does not seem to be a major mechanism of anti-parental tumor immunity induced by xenogenized variant cells.     

DNA Repair, H-2, and Aging in NZB and CBA Mice

Current evidence suggests that a correlation exists between the capacity to perform excision repair of UV-induced DNA damage and maximum lifespan in different species. Preliminary evidence has also indicated differences of DNA repair capacities in lymphocytes of several strains of mice congenic at the H-2 locus. It is known that the H-2 system influences maximum lifespan potential in mice. In the present studies excision repair of UV-induced DNA damage, but not gamma-induced damage, was found to correlate with mean survival in the adult inbred mouse strains NZB and CBA, using PHA stimulated splenic lymphocytes. Furthermore, in (NZB × CBA)F₂ hybrid adult progeny the level of DNA repair of UV-induced damage corresponded to the H-2 allele (H-2^d/2^d from NZB or H-2^k/2^k from CBA) inherited from the parental strain. These studies suggest the possibility of a tricornered relationship between the main histocompatibility complex, one form of DNA repair, and lifespan within the species.     

EXPERIMENTAL ACTIVATION OF LATENT CYTOMEGALOVIRUS INFECTION OF THE CAPTIVE BRED (F1) CYNOMOLGUS MONKEYS BY LIVE OR KILLED VARICELLA-ZOSTER VIRUS INOCULATED UNDER IMMUNOSUPPRESSION

F₁ cynomolgus monkeys bred in captivity and thought to be "SPF" had latent cytomegalovirus (CMV) infection although less frequently than in wild-born monkeys. Latent CMV could be activated under severe immunosuppression by Inoculation with a certain strain of varicella-zoster virus (VZV) as shown in a previous study.¹ We carried out further experiments using live and formalin-inactivated VZV in F₁ monkeys which were sero-positive for CMV, but not for any other viruses. The results showed that both live and inactivated VZV were equivalent in permitting activation of latent CMV, suggesting that the VZV inoculum merely played an immunogenic, but not any virological, role in this case. Captive F₁ monkeys, however, carried fewer CMV, and/or resisted CMV reactivation more than the wild monkeys in the previous studies,¹ judging from the time required for generalized CMV infection to become expressed. ACTA PATHOL JPN 38: 967∼978, 1988.     

Transgenerational effects of prenatal nutrient restriction on cardiovascular and hypothalamic-pituitary-adrenal function

The perinatal environment is a powerful determinant of risk for developing disease in later life. Here, we have shown that maternal undernutrition causes dramatic changes in heart structure and hypothalamo-pituitary-adrenal (HPA) function across two generations. Pregnant guinea pigs were fed 70% of normal intake from gestational days 1–35 (early restriction; ER), or 36–70 (late restriction; LR). Female offspring (F₁) were mated and fed ad libitum to create second generation (F₂) offspring. Heart morphology, blood pressure, baroreceptor and HPA function were assessed in male F₁ and F₂ offspring. ER_F1 males exhibited elevated blood pressure, increased left ventricular (LV) wall thickness and LV mass. These LV effects were maintained in the ER_F2 offspring. Maternal undernutrition increased basal cortisol and altered HPA responsiveness to challenge in both generations; effects were greatest in LR groups. In conclusion, moderate maternal undernutrition profoundly modifies heart structure and HPA function in adult male offspring for two generations.     

Immunohistochemical Investigation of Vimentin, Neuron-specific Enolase, α1-antichymotrypsin and α1-antitrypsin in Adenoid Cystic Carcinoma of the Salivary Gland

Fifty-four adenoid cystic carcinomas (ACC) arising in major and minor salivary glands as well as in normal salivary glands were studied by immunohistochemistry for the presence of vimentin, neuron specific enolase (NSE), α₁-antichymotrypsin (α₁-ACT) and α₁-_- antitrypsin (α₁-_- AT). Five patterns of histological differentiation were found in ACC, and for the cellular components of each, it was possible to establish a special immunohistochemical profile. In ACC, vimentin-positive cells were observed in the outer tubular, cyst-lining and small angular cells. NSE was positive in the myoepithelial cells of normal salivary gland. Neoplastic cells of ACC showed NSE positivity mainly in the small angular cells and partly in the duct luminal cells. α₁-ACT was localized in the intercalated duct cells and serous acinar cells of normal salivary gland, and in the duct luminal cells of ACC. α₁-AT could not be detected in any of the epithelial cells of normal salivary gland. In ACC, eosinophilic hyaline material in the cribriform spaces was positive for α₁-AT, but no positivity was demonstrated in tumor cells. The present study showed that there are at least two populations of tumor cells in ACC: duct luminal cells that express α₁-ACT, thus indicating their ductal character, and small angular cells that express vimentin, characteristic of non-luminal cells. Moreover, our results indicate that α₁-AT is a useful marker of basement membrane-like material.