Wayne S. Lapp

Expression of Tumor Necrosis Factor Alpha in the Developing Nervous System

We present evidence that tumor necrosis factor alpha (TNF-alpha) is transiently expressed at specific times during embryogenesis in precisely defined areas of the nervous system in two different classes of vertebrates. In murine embryos, TNF-alpha was detected in the brain, neural tube and peripheral mixed spinal nerves. In the chick embryo, TNF-alpha was observed in the brain neuroepithelium and in the developing Purkinje neurons of the cerebellum. Western immunoblot analysis revealed that brain tissue from both mouse and chick embryos contained a 50 kDa protein showing immunoreactivity with anti-TNF-alpha antibody. These results suggest that TNF-alpha participates in the normal development of the vertebrate brain and spinal cord.

T cell functional defect associated with thymic epithelial cell injury induced by a graft-versus-host reaction.

SUMMARY The injection of parental strain cells into adrenalectomized (CBA x A)F1 mice induced a graft-versus-host (GVH) reaction which was morphologically characterized by thymus epithelial cell injury but not stress-related thymic involution. Thymocytes from mice undergoing a GVH reaction were studied for their ability to reconstitute allograft reactivity in thymectomized, irradiated, bone marrow reconstituted (ATxBM) (CBA X A)F1 mice. Thymocytes of mice experiencing a GVH reaction were &thetas;-positive during the course of the reaction, however, by 40 days after GVH induction these thymocytes were unable to reconstitute allograft reactivity to H-2-incompatible skin grafts. It is proposed that GVH-induced thymic epithelial cell injury prevents or arrests normal T cell differentiation, resulting in a population of thymocytes which lack complete functional capability.

Precocious thymic involution manifest by epithelial injury in the acquired immune deficiency syndrome.

Thymuses from six heterosexual Haitian patients with the acquired immune deficiency syndrome (AIDS) were studied by light microscopy and the findings were compared with those from three control groups. The control groups included 1) five age-matched Haitian hospital patients; 2) ten age- and sex-matched Montreal patients who had died suddenly or had had brief illnesses; and 3) 20 middle-elderly Montreal patients who had experienced chronic, wasting illnesses or prolonged hospitalization. Thymuses from patients with AIDS demonstrated pronounced involution, effacement of the cortex and medulla, marked thymocyte depletion, variable degrees of plasma cell infiltration and fibrosis, and, above all, absence of Hassalls corpuscles. Thymuses from Haitian and Montreal control subjects who had died suddenly or had brief illnesses demonstrated minimal involution and abundant Hassalls corpuscles. Although thymuses from 12 of the chronically ill control subjects demonstrated marked involution, architectural effacement, and absence of Hassalls corpuscles, partial architectural preservation and variable numbers of Hassalls corpuscles were observed in eight of these subjects. Thus, the extent of thymic involution observed in patients with AIDS antedates that incurred with aging and supersedes that induced by sustained stress and inanition. The loss of Hassalls corpuscles in patients with AIDS suggests that the thymic epithelium either incurs a form of injury or undergoes precocious involution during the illness. Whether this lesion is central to the pathogenesis of AIDS or merely a reflection of intense, sustained stress coupled with accelerated physiologic involution is unknown. It is possible that the disappearance of Hassalls corpuscles may indicate important, although as yet cryptic events within the thymic microenvironment in this syndrome.

Graft-versus-host reactions in the beige mouse: an investigation of the role of host and donor natural killer cells in the pathogenesis of graft-versus-host disease

To investigate the role of natural killer (NK) cells in the induction and pathogenesis of graft-versus-host (GVH) disease, +/beige (+/bg; normal NK cell activity) and beige/beige (bg/bg; deficient NK cell activity) parental C57BL/6 (B6) lymphoid cells were used to induce GVH reactions in either B6xC3H/Hej +/bg (+/bgF1) or B6°C3H/HeJF1 bg/bg (bg/bg F1) hybrid mice. When B6 bg/bg parental lymphoid cells (PLC) were injected into bg/bg F1 mice, early splenomegaly, early severe suppression of the plaque-forming cell (PFC) response to sheep red blood cell (SRBC), and only partial suppression of T cell mitogen responses to concanavalin A (Con A) and phytohemagglutinin (PHA) were observed on day 12 after GVH induction. In the same GVH combination, slightly augmented NK cytotoxic activity was induced and no GVH-induced moderate-to-severe pathological alterations in the liver and pancreas were observed. When bg/bg PLC were injected into +/bg F1 mice, early splenomegaly and pronounced immunosuppression of the PFC response to SRBC and partial suppression of Con A and PHA responses were observed on day 12 after GVH induction. In this combination (bg/bg +/bg F1), significant NK cell activity was induced, but no moderate-to-severe histopathological alterations were observed. In contrast, when B6 +/bg PLC were injected into either +/bg F1 or bg/bg F1 hybrids, early splenomegaly, and severe immunosuppression of both the PFC response to SRBC and the T cell mitogen responses to Con A and PHA were observed by day 12—which persisted until day 30 after GVH induction. Furthermore, high NK cell activity was recorded and moderate-to-severe histopathological alterations appeared in both +/bg F1 and bg/bg F1 recipients. These results show that the bg/bg PLC can induce GVH-associated early splenomegaly and immunosuppression of the PFC response to SRBC in both the bg/bg F1 and +/bg F1 hybrids, but that it failed to induce moderate-to-severe histopathological alterations, even though NK cell activity of host origin was activated during GVH reactions. Conversely, when +/bg donor cells were used to induce the GVH reaction, splenomegaly and immunosuppression, as well as moderate-to-severe histopathological lesions were induced. These results suggest that donor NK cells rather than host NK cells play an active role in GVH-associated tissue damage, which in turn contributes to the long-term suppression.

The graft-versus-host reaction and immune function. I. T helper cell immunodeficiency associated with graft-versus- -host-induced thymic epithelial cell damage.

The injection of parental A strain lymphoid cells into adrenalectomized CBAxA F1 (BAF1) mice induced a chronic graft-versus-host (GVH) reaction resulting in T cell and B cell immunosuppression as well as thymic epithelial cell injury, but not stress-related thymic involution. Thymocytes from BAF1 mice undergoing a GVH reaction were studied for their ability to reconstitute T helper cell (TH) function and phytohemagglutinin (PHA) and concanavalin A (Con A) mitogen responses in thymectomized, irradiated, BAF1 mice reconstituted with normal syngeneic bone marrow (ATxBM). Thymocytes from BAF1 mice early after the induction of a GVH reaction (days 10-12) were as effective as normal thymocytes in reconstituting TH and mitogen responses. Thymocytes from BAF1 mice 40 or more days after the induction of a GVH reaction did not reconstitute either the TH function or PHA and Con A responses in ATxBM mice. The inability to reconstitute ATxBM mice was not due to the presence of suppressor cells contained in the thymocyte inoculum. It is proposed that GVH-induced thymic epithelial cell injury blocks or arrests normal T cell differentiation, resulting in a population of thymocytes that lack the potential to become competent T helper cells or mitogen-responsive cells when transferred into ATxBM mice. This thymic functional defect results in a permanent TH immunodeficiency in mice experiencing a chronic GVH reaction.

PROGRESSIVE ACCUMULATION OF BACTERIAL LIPOPOLYSACCHARIDE IN VIVO DURING MURINE ACUTE GRAFT-VERSUS-HOST DISEASE

In a previous report the authors demonstrated that acute graft‐versus‐host disease (GVHD) was associated with pathologic amounts of tumour necrosis factor alpha (TNF‐α) and the appearance of lipopolysaccharide (LPS) in the blood of GVH reactive mice just prior to death. In this study the authors have investigated the kinetics of LPS accumulation in different organs during the course of acute GVHD using a murine model. Unirradiated C57BL/6×AF1 (B6AF1) mice were transplanted with C57BL/6 (B6) lymphoid cells and killed at predetermined times after transplantation for LPS analysis. Control animals were injected with either 60×106 B6AF1 lymphoid cells (syngeneic) or 60×106 irradiated (2000 rad) CBA lymphoid cells (allogeneic). Lipopolysaccharide began to appear in the liver and the spleen of GVH reactive mice from day 2 post‐transplant and by day 10 all GVH reactive mice tested positive for hepatic and splenic LPS. Low levels of LPS were detected in some control mice from days 2 to 10 post‐transplant but LPS was never detected after day 10 in control groups. Total hepatic and splenic LPS in acute GVH reactive mice peaked at a time coincident with the appearance of LPS in the serum and with the onset of mortality. These results demonstrate that tissue levels of LPS increase throughout the course of acute GVHD and are sufficient to trigger the release of pathologic amounts of TNF‐α from primed macrophages resulting in the cachexia and mortality associated with acute GVHD in this model.

T cell subsets in the thymus of graft-versus-host immunosuppressed mice. Sensitivity of the L3T4+Lyt-2- subset to cortisone.

We have investigated the effects of graft-versus-host disease on T cell differentiation in the murine thymus. We previously reported that GVH-induced thymic dysplasia results in a T cell immunodeficiency associated with a lack of IL-2 production. This deficiency in IL-2 production may be the result of a reduction in the number of L3T4+Lyt-2- IL-2-producing cells or of a functional defect in this population. To test these two alternatives, flow cytometry analysis of L3T4 and Lyt-2 antigen expression on thymocytes along with immunofluorescence microscopy were employed to assess T cell phenotypes in thymuses of GVH mice. GVH reactions were induced by injecting 40 x 10(6) C57BL/6 (B6) or A strain lymphoid cells into C57BL/6xAF1 (B6AF1) mice. Thymocyte populations were quantitated on different days after GVH induction. In the normal thymus, the ratio of L3T4/Lyt-2 single positive cells was greater than 2:1. In contrast, such a ratio was less than 1:1 in the atrophic GVH thymus, owing to a selective reduction in the number of L3T4+Lyt-2- cells. Following cortisone treatment the ratio of L3T4/Lyt-2 single positive thymocytes in normal F1 mice was approximately 3:1, whereas in GVH animals this ratio was reversed (1:2). This reversal was due to a selective reduction in the absolute numbers of L3T4+Lyt-2- cells. In adrenalectomized GVH animals, thymic cortical atrophy was prevented and normal ratios of L3T4/Lyt-2 single positive cells were observed. However, when these animals were treated with cortisone, the L3+T4/Lyt-2- population was more sensitive than was the L3T4- Lyt2+ population, thereby resulting in a 1:2 L3T4/Lyt-2 ratio. These results demonstrate that single positive L3T4 cells are present in the murine GVH thymus, yet they have not acquired cortisone resistance, a trait normally attributed to this mature thymic subset. It appears that the GVH dysplastic thymus can support the differentiation of L3T4+Lyt-2- cells--however, such a thymus is unable to confer cortisone resistance upon this population. Consequently, these cells appear to be eliminated when exposed to corticosteroids in peripheral lymphoid tissue.

Graft versus host-induced immunosuppression: mechanism of depressed T-cell helper function in vitro.

Abstract The cellular basis of graft versus host (GVH)-induced immunosuppression was investigated. Results showed that thymus, lymph node, and splenic T cells from normal mice and thymus and lymph node T cells from GVH mice, when cultured on one side of a cell impermeable membrane, restored the plaque-forming cell (PFC) response to sheep erythrocytes of GVH-immunosuppressed spleen cells (GVH-SC) cultured on the other side of the membrane. The restoring ability of T cells present in GVH-SC was inhibited by splenic accessory (A) cells. A direct relationship was shown between the proportion of splenic A cells and the degree of suppression of the PFC response during the first 10 days of the GVH reaction. Normal or GVH A cells reconstituted the PFC response of normal cells and GVH-SC depleted of their A-cell fraction. An optimum ratio of A: nonadherent (NA) cells (1: 10) was required for maximum reconstitution. Larger proportions of A cells inhibited the PFC response. The results suggest that GVH-induced immunosuppression is due, at least in its initial phase, to a depressed T-cell helper function caused by a marked increase of A cells in the spleen.

Graft-versus-host induced immunosuppression: Depressed T cell helper function in vitro☆

Abstract The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.

KINETICS OF NATURAL KILLER CELLCYTOTOXICITY REACTION: Relationship Between Natural Killer Cell Activity, T and B Cell Activity, and Development of Histopathological Alterations

The relationships between splenic natural killer (NK) cell cytotoxicity, T and B cell function, and the development of histopathological lesions in the liver and pancreas have been studied during the course of graft-versus-host (GVH) reactions. GVH reactions were induced in (C57BL/6A)F1 (B6AF1) hybrids by different doses, (10,20 and 30×106) of either parental strain C57/BL6 (B6) or A lymphoid cells. Splenic NK cell cytotoxicity was studied by employing YAC-1, an NK-eellsensitive target. Splenic T and B cell function were assessed by mitogen responsiveness to concanavalin A, phytohemagglutiain, and Escherichia coli lipopolysaccharide, and by the in vitro plaque-forming cell response to sheep red blood cells. Histopathological lesions characteristic of GVH reactions were recognized at a time (day 8 after GVH induction) when both T and B cell functions were totally suppressed and NK cell activity was greatest. The severity of histopathological alterations later (day 16 after GVH induction) correlated with an early peak in NK cell cytotoxicity rather than with the overall NK cell activity. When low doses (10,20×106) of B6 cells were employed to induce GVH reactions, a significant increase in NK cell activity was observed, yet neither histopathological alterations nor suppression of T and B cell functions were observed. The killing of YAC-1 targets by splenocytes obtained from the different GVH combinations could not be abrogated by pretreatment with anti-Thy 1.2 serum plus complement, suggesting that T lymphocytes were not central to this cytolytic process. These experiments demonstrated that: (1) an inverse relationship between T and B cell function and NK cell activity was observed early after GVH induction, (2) the severity of histopathological lesions and immunosuppression, as well as the degree of overall augmented NK cell activity, was determined by the dose and genotype of donor cells injected to induce GVH reactions, and (3) GVH-associated moderate-severe lesions occurred only in groups in which NK cell activity peaked early—-whereas-when NK cell activity peaked later, either mild or no lesions were observed, suggesting that the early rapid increase of NK cell activity may be useful for predicting the severity of GVH pathogenesis.