Macrophages perform both injury-inducing and repair-promoting tasks in different models of inflammation, leading to a model of macrophage function in which distinct patterns of activation have been proposed. We investigated macrophage function mechanistically in a reversible model of liver injury in which the injury and recovery phases are distinct. Carbon tetrachloride-induced liver fibrosis revealed scar-associated macrophages that persisted throughout recovery. A transgenic mouse (CD11b-DTR) was generated in which macrophages could be selectively depleted. Macrophage depletion when liver fibrosis was advanced resulted in reduced scarring and fewer myofibroblasts. Macrophage depletion during recovery, by contrast, led to a failure of matrix degradation. These data provide the first clear evidence that functionally distinct subpopulations of macrophages exist in the same tissue and that these macrophages play critical roles in both the injury and recovery phases of inflammatory scarring.

Background & Aims: Liver fibrosis and cirrhosis result from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs). Previously considered irreversible, we have studied a model of cirrhosis to determine the mechanisms mediating and limiting spontaneous recovery. Methods: A micronodular cirrhosis was induced in rats after 12 weeks of CCl4 intoxication. Livers were analyzed for evidence of matrix degradation, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression, stellate cell apoptosis, tissue transglutaminase (tTg) expression, and matrix cross-linking during spontaneous recovery of up to 366 days. Results: Over 366 days of recovery, micronodular cirrhosis underwent significant remodeling to a macronodular cirrhosis. Expression of collagen-1 and TIMP messenger RNA (mRNA) decreased significantly and active MMPs were shown in livers during remodeling of fibrosis. Resolution also was characterized by apoptosis of HSCs, predominantly at the margins of fibrotic septa. Residual septa, not remodeled at 366 days, were characterized by tTg-mediated cross-linking and relative hypocellularity. Conclusion: Recovery from comparatively advanced cirrhosis is possible and results in remodeling from a micronodular cirrhosis to a macronodular cirrhosis. We suggest resolution is limited by tTg-mediated matrix cross-linking and a failure of HSC apoptosis.

A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.

BACKGROUND:Conflicting data on the effect of the Epstein-Barr virus (EBV) on outcome in Hodgkin's disease (HD) might be due to the heterogeneous nature of this disease. In this study we have investigated whether the effect of EBV status on outcome is different between aetiologically defined age groups (15-34, 35-44, 45+ years) and also between males and females. PATIENTS AND METHODS:Paraffin-embedded sections from 273 patients with advanced HD from two related clinical trials were analysed for the presence of EBV using in situ hybridisation. RESULTS:EBV was detected in 78 (29%) of cases. For all patients, after a median follow-up of 5 years, there were no significant differences in survival by EBV status although there was a trend towards longer failure-free survival times for EBV-positive patients. Multivariate analyses suggested that EBV and sex, when in combination, were prognostic factors for failure-free survival (P = 0.06 for both). For subgroups, the effect of EBV on failure-free survival was significant for males and 15-34 years age group (P = 0.05 and P = 0.03, respectively). CONCLUSION:This study suggests that with a median follow-up of 5 years, EBV status does not affect survival but being EBV-positive may be beneficial in terms of failure-free survival, particularly for males and younger adults.

A number of studies in developed countries have reported variation in the frequency of Epstein-Barr virus (EBV)-associated Hodgkin's disease (HD) with age. A 'three disease' model for HD that incorporates the EBV association, histological subtype of HD and age has recently been proposed. In this model, Hodgkin's disease of childhood and older adults is commonly EBV-associated and of mixed cellularity type, whereas young adult HD is generally not EBV-associated and is usually characterised by nodular sclerosis disease. A case series of HD diagnosed in the West Midlands between 1981 and 1997, inclusive, was used to investigate the applicability of the 'three disease' model. In situ hybridisation for the EBV early RNAs (EBERs) was used to determine the presence of EBV in the malignant Hodgkin/Reed-Sternberg cells. In contrast to the 'three disease' model, nodular sclerosis was the predominant subtype in each of the age groups within the case series. In addition, overall there was little variation in EBV-positive rates across the age ranges examined. However, when females were analysed separately, older women (45+ years) were significantly more likely to have EBV-positive disease than their younger counterparts (<45 years). In summary our results do not generally support the 'three disease' model.

We have analyzed paraffin sections from 190 patients with histologically confirmed Hodgkin’s disease (HD) for the presence of Epstein-Barr virus (EBV) using in situ hybridization to detect the EBV-encoded Epstein-Barr virus early RNAs (EBERs) and immunohistochemistry to identify latent membrane protein-1 (LMP1) expression. EBV was present in the tumor cells in 51 HD cases (27%) and was mainly confined to the mixed cellularity and nodular sclerosis subtypes. There was no difference between EBV-positive and EBV-negative HD patients with regard to age, clinical stage, presentation, and the number of alternating chemotherapy cycles of ChIVPP and PABIOE received. The complete remission rate after study chemotherapy was 80% in EBV-positive patients versus 69% in EBV-negative patients (P = .05). The 2-year failure-free survival rate was significantly better for EBV-positive patients when compared with the EBV-negative HD group (P = .02). Although 2-year and 5-year overall survival rates were better for EBV-positive HD patients, the differences were not statistically significant (P = .18 andP = .40, respectively). In conclusion, the results confirm the favorable prognostic value of EBV in the tumor cells of HD patients and suggest important differences in response to chemotherapy between EBV-positive and EBV-negative patients.

Approximately 40% of Hodgkin's disease (HD) cases in Western countries carry Epstein-Barr virus (EBV) in the malignant Hodgkin-Reed-Sternberg (H-RS) cells. HLA class I–restricted cytotoxic T lymphocytes (CTLs) with specificity for viral antigens expressed in H-RS cells therefore have therapeutic potential. However, a prerequisite for CTL therapy is that the tumor target be capable of processing and presenting endogenously expressed antigens via the transporter associated with antigen processing (TAP)-dependent HLA class I pathway. We have assessed the antigen-presenting phenotype of H-RS cells in two ways. First, immunohistochemical analysis of 38 HD biopsies showed that H-RS cells were uniformly TAP1/TAP2-positive and expressed HLA class I in the majority (18 of 24, 75%) of EBV-positive cases compared with only 4 of 14 (29%) of EBV-negative cases. Second, using a panel of 5 H-RS cell lines, we showed that 4 of 5 could process and present EBV proteins to HLA class I–restricted EBV-specific CTL clones. Others have reported that human interleukin-10 (IL-10), which is expressed by H-RS cells in the majority of EBV-positive HD cases, can abrogate CTL recognition in some circumstances. However, IL-10 pretreatment of the H-RS lines or of the EBV-specific CTLs had no such effect in this system. These results support the possibility that EBV-specific CTLs may be used to treat virus-positive HD. © 1998 by The American Society of Hematology.

The Epstein-Barr virus (EBV)-encoded latent membrane proteins, LMP1 and LMP2, are consistently expressed by the malignant Hodgkin/Reed-Sternberg (HRS) cells of EBV-associated Hodgkin's disease (HD). Cytotoxic T lymphocyte (CTL) responses to both of these proteins have been shown in the blood of EBV-seropositive individuals, yet in HD the apparent failure of the CTL response to eliminate HRS cells expressing LMP1 and LMP2 in vivo has given rise to the suggestion that HD may be characterized by the presence of defects in antigen processing/presentation or in CTL function. This study has used immunohistochemistry to show high-level expression of major histocompatibility complex (MHC) class I molecules by the HRS cells of EBV-associated HD and either low level or absence of expression of MHC class I molecules on HRS cells of EBV-negative tumors. In addition, HRS cells expressed high levels of transporter-associated proteins (TAP-1, -2), irrespective of the presence of latent EBV infection. These results suggest that global downregulation of MHC class I molecules does not account for the apparent ability of EBV-infected HRS cells to evade CTL responses, but may be important in the understanding of EBV-negative disease.We have also sequenced an epitope in LMP2A (CLGGLLTMV) that is restricted through HLA A2.1, a relatively common allele in Caucasian populations, and showed that this epitope is wild type in a small group of EBV-associated HLA A2.1-positive HD tumors. This result may be relevant to proposed immunotherapeutic approaches for EBV-positive HD patients that target CTL epitopes.

Posttransplantation lymphoproliferative disease (PTLD) is virtually always associated with Epstein-Barr virus (EBV) infection. BCL-2 and other proteins that confer resistance to apoptosis have been implicated in the pathogenesis of a variety of malignancies including lymphomas. One EBV protein, BHRF1, is a homologue of BCL-2, whereas another, the latency membrane protein 1 (LMP-1), upregulates BCL-2 expression in vitro. In the present study, we used immunohistochemistry to study the expression of these viral and cellular proteins as well as a variety of other EBV-encoded proteins in PTLD. BHRF1 was not detected in any PTLD specimen, whereas BCL-2 was shown in 12 of 17 lesions examined. With one exception, all LMP1-positive cases also expressed BCL-2 and the absence of LMP1 was always associated with a lack of BCL-2 expression. The results do not support a role for the EBV homologue of BCL-2 in PTLD, but they do support a role for viral induction of BCL-2 expression.