The duplicitous effects of interleukin 4 on tumour immunity: how can the same cytokine improve or impair control of tumour growth?

Successful tumour immunity relies on innate and adaptive immune responses, with cytokines like interleukin 4 (IL-4) known to influence tumour clearance in both positive and negative ways. Here, we summarise some of the murine tumour models used over the past two decades to assess the impact of IL-4 on tumour immunity, with emphasis on the effects of IL-4 on the tumour-induced CD8 T-cell response. These data are compared with our own recent studies showing that IL-4 impairs CD8+ T-cell-mediated immunity against the mastocytoma cell line P815 expressing the immunogenic HLA-CW3 gene; moreover, we hypothesise that quantitative and qualitative differences in the HLA-CW3-induced CD8+ T-cell response impair control of tumour growth and aid the development of secondary tumours. We conclude that the duplicitous effects of IL-4 on tumour immunity depend on the type of effector cell (adaptive/innate) mediating tumour clearance and whether tumour growth depends on stromal infrastructure. Thus, the search for factors that improve or weaken the effectiveness of tumour-specific T cells has to be continued to improve modern approaches of immunotherapy against cancer.

Persistence of donor-reactive CD4+ T cells in liver and spleen of rats tolerant to a liver allograft.

BACKGROUND: In the rat, orthotopic liver transplantation from a DA strain donor to a PVG recipient causes an early rejection response that spontaneously resolves over the following weeks to yield long-lasting, donor-specific tolerance. METHODS: Limiting dilution analysis was used to estimate the frequencies of host CD4+ cells able to proliferate in response to donor antigens in the grafted liver and spleen of recipients during and after tolerance induction. RESULTS: Compared with naive PVG rats, both the frequencies and absolute numbers of donor-reactive host CD4+ cells in the liver and spleen rose significantly during the first week after transplantation and remained elevated for at least 3 months. CONCLUSION: We conclude that the development of tolerance in this model is not associated with deletion of clonogenic donor-reactive CD4+ T cells by clonal exhaustion or any other mechanism.

Contrasting phenotypes of liver-infiltrating leucocytes isolated from MCMV-infected BALB/c and C57BL/6 mice.

We characterized liver-infiltrating leucocytes (LIL) from BALB/c and C57BL/6 mice 0-56 days after murine cytomegalovirus (MCMV) infection. Inflammation clears from C57BL/6 mice 4-5 weeks post infection (pi), but persists for several months in BALB/c mice. The LIL obtained were 60-80% Thy 1.2+ by flow cytometry. The percentage of CD8+ cells rose sharply in all mice 7 days pi, with little decrease in BALB/c mice by day 56. CD4-CD8-Thy 1.2+/TCR alpha beta + cells were more prevalent in LIL than lymph node cells (LNC) irrespective of MCMV infection, whilst infection increased the proportion of CD8+ L-selectin- LIL (but not LNC). LIL from both mouse strains demonstrated cytotoxic activity against YAC-1 cells, but only LIL from BALB/c mice proliferated spontaneously ex vivo 21 days pi, as measured by tritiated thymidine incorporation. BALB/c LIL produced IFN gamma and IgG2a 7-21 days pi, whilst IL-10 secretion was similar in both strains. Thus, persistent hepatitis in BALB/c mice is associated with activation and proliferation of intrahepatic leucocytes with some bias towards a Th1 response.

The association between murine cytomegalovirus induced hepatitis and the accumulation of oval cells.

The accumulation of oval cells is an early event in the development of hepatocellular carcinoma induced by certain experimental regimes involving hepatocarcinogens. Oval cells have also been observed during chronic hepatitis induced by alcohol and iron overload. In this study, livers of murine cytomegalovirus (MCMV) infected mice were examined to determine whether hepatitis induced by this virus could initiate oval cell proliferation. BALB/c and C57BL/6 mice were infected with MCMV and studied 4, 8, 10 and 12 months later, alongside control (uninfected) mice. The livers were examined histochemically, immunocytochemically and by in situ hybridization to identify oval cells, inflammatory cells and proliferating cells. Oval cells were seen in the periportal regions of livers from MCMV infected BALB/c mice. These increased in number from 4 to 12 months after infection in parallel with increases in the numbers of inflammatory cells, even though cells expressing MCMV antigens were no longer evident in these samples. Proliferating oval cells and hepatocytes were identified by PCNA staining, indicating an increased level of liver regeneration in the infected livers. C57BL/6 mice are less susceptible to persistent MCMV hepatitis and had fewer oval cells than BALB/c mice. Thus the study demonstrates an association between MCMV induced hepatitis, inflammation, and presence of oval cells.

The roles of tumour necrosis factor-alpha, interleukin-1 and interleukin-12 in murine cytomegalovirus infection.

The roles of the inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-12, in murine cytomegalovirus (MCMV) disease were investigated in susceptible BALB/c and resistant C57BL/6 mice. MCMV infection induced IL-1 and TNF-alpha production by peritoneal cells from BALB/c mice, as demonstrated previously in C57BL/6 mice. Overt ill-health and viral replication in the spleens of BALB/c mice were increased by in vivo treatment with soluble TNF-alpha receptors to inhibit the activity of this cytokine, whilst antibodies to IL-12 had a similar but more restricted effect C57BL/6 mice were not affected by either treatment, suggesting TNF-alpha and IL-12 are not critical for natural killer cell-mediated restriction of viral replication in the spleen. Soluble TNF-alpha receptors and antibodies to IL-12 also enhanced MCMV titres and numbers of viral antigen-positive cells in the livers of BALB/c mice and TNF-alpha receptors have similar effects in C57BL/6 livers. In contrast, IL-1 receptors improved the health of MCMV-infected BALB/c mice and reduced viral replication and hepatitis at some time-points. Mechanisms which may underlie these changes are discussed.

Effect of a retroviral immunodeficiency syndrome on murine cytomegalovirus-induced hepatitis.

We have studied the effects of LP-BM5 MuLV-induced murine acquired immunodeficiency syndrome (MAIDS) on concomitant murine cytomegalovirus (MCMV) infection in the livers of C57BL mice. A delayed inflammatory response in livers of mice with MAIDS (M+) on day 4 was associated with impaired clearance of MCMV-infected cells 6 days after infection. This correlated with increased levels of inflammation and serum alanine transaminase. The latter reflects enhanced hepatic necrosis, which was evident histologically. Delayed-type hypersensitivity responses to MCMV antigen were unimpaired in M+ mice and were mediated by CD8+ cells. Depletion of NK1.1+ cells from M+ mice increased MCMV replication and associated liver damage on day 6, whereas CD8+ depletion had little effect. In contrast, in the presence of CD8+ cells M- C57BL mice did not require NK1.1+ cells for control of hepatic MCMV infection, but dual NK1.1+ and CD8+ depletion dramatically potentiated hepatic MCMV replication. Our results suggest that M+ mice may acquire non-NK1.1+ and non-CD8+ cells that are able to partially control hepatic MCMV infection. These findings are discussed with reference to mortality in M+ mice after high-dose MCMV infection, as this is initially delayed but ultimately higher than in M- controls.

Adrenalitis and the adrenocortical response of resistant and susceptible mice to acute murine cytomegalovirus infection.

Murine cytomegalovirus (MCMV) induces adrenalitis in BALB/c mice but does not compromise adrenal function, assessed by levels of circulating adrenocorticotropic hormone (ACTH) and by the response to challenge with synthetic ACTH. Levels of corticosterone increased 2 days after infection in mice of this strain, consistent with previously established interactions between mediators of acute inflammation and activation of the hypothalmus-pituitary-adrenal axis. Moreover, an adrenocortical response was critical to survival of BALB/c (but not C57BL/6) mice 3 days after infection, as pharmacologically or surgically adrenalectomized BALB/c mice died when given doses of virus up to fivefold lower, than they could normally tolerate. However, death could not be prevented by the administration of soluble cytokine receptors to inhibit the action of interleukin 1 (IL-1) or tumour necrosis factor alpha (TNF alpha). The corticosteroid response did not mediate.MCMV-induced thymic atrophy. As the above traits were all less evident in C57BL/6 mice, a common genetic basis is discussed.

Cytomegalovirus hepatitis: characterization of the inflammatory infiltrate in resistant and susceptible mice.

Mice susceptible and resistant to murine cytomegalovirus (MCMV) were infected with this virus and livers were harvested after 2-231 days. Cryostat sections were stained to visualize cells bearing CD4, CD8 or Mac-1 antigens. Mac-1+ cells were prevalent in inflammatory foci after 2 days. These cells persisted in susceptible BALB/c and A/J mice, but disappeared from livers of resistant C57Bl/6 and CBA/CaH mice by day 28. T cell inflammation peaked on days 7-11. This declined by day 56 in C57Bl/6 and CBA/CaH mice, but persisted in BALB/c and A/J mice for at least 231 days. Persistent CD8+ cells were dispersed throughout the parenchyma. More CD8+ cells were observed 7-14 days after infection in the livers of bg/bg (natural killer (NK) cell-deficient) C57Bl/6 and CBA mice, and in C57Bl/6 mice depleted of NK1.1 cells by MoAb. Thus, mice of strains susceptible to MCMV exhibit hepatitis characterized by persistence of dispersed CD8+ cells. This phenomenon may be limited by NK cells in resistant strains.

Characterization of thymic involution induced by murine cytomegalovirus infection.

Infection of BALB/c mice with murine cytomegalovirus (MCMV) decreased the numbers of cells recovered from the thymus by 80-90% after 4-7 days, although less than 10 thymocytes per million were productively infected with the virus. A loss of cortical thymocytes was evident in histologic sections and correlated with depletion of CD4+ CD8+ cells. Thymic involution was minimal in C57BL/6 mice. This resistance was not H-2b-associated, as BALB.B (H-2b) mice were severely affected. In CXB recombinant inbred mice, thymic involution and MCMV replication co-segregated with atrophy and infection of the spleen and bone marrow. This suggests common regulation by natural killer (NK)1.1+ cells, consistent with the enhanced thymic involution demonstrated in NK-deficient bg/bg mice. However, CD4- CD8- cells were not depleted, so bone marrow hypoplasia may not be the proximal cause of thymic involution. MCMV infection activated CD4+, CD8+ and CD4+ CD8+ thymocytes, as expression of MEL14, major histocompatibility complex class I (H-2) and Sca-1 antigens increased on these cells. In vitro lymphoproliferation and interleukin (IL)-3 release were enhanced in unseparated and CD4(+)-enriched thymus preparations. Maturation of the thymus population was also evident from the high frequencies of single positive CD4+ and CD8+ cells and the decline in Sca-2 expression. However, unlike peripheral T cells, thymocytes from infected mice did not release IL-2. The results suggest that thymic involution accelerates the transit of cells through the thymus. The possibility that this impairs the elimination of autoreactive T cells within the thymus and promotes the autoimmune manifestations of MCMV disease is discussed.

B-cell activation following murine cytomegalovirus infection: implications for autoimmunity.

Infection of susceptible mice with murine cytomegalovirus (MCMV) induces persistent inflammation, and the production of autoantibodies reactive with large numbers of proteins from all major organs. However the roles of polyclonal B-cell activation, autoreactive T-helper cells and host-virus cross-reactions in these phenomena have not been evaluated. The present study reveals six- to 20-fold increases in serum immunoglobulin levels in MCMV-infected BALB/c and CBA mice, with IgG3 and IgG2b most affected. Titres of antibodies reactive with autologous tissues and ovalbumin (OVA) also increased following MCMV infection, whilst responses to a synthetic antigen [polyvinyl pyrrolidone (PVP)] were unaffected or depressed. IgG2a was the isotype most affected in responses to OVA, MCMV antigens and autologous tissues, suggesting interferon-gamma (IFN-gamma) may contribute to responses induced in the presence of the relevant antigen. Increases in total and antigen-specific immunoglobulin levels were CD4 dependent, as they were reduced in infected mice depleted of these cells with anti-CD4 antibodies. Serological changes were preceded by B-cell expansion and activation evident from increased cell yields, frequencies of cells releasing immunoglobulin and proliferation of T-depleted spleen and lymph node preparations. Numbers of mature B cells and macrophages increased in the lymph nodes, but B-1a (CD5+ Ig+) cell counts remained low. Alterations in the B-cell phenotypic profiles were more complex in the spleen, but correction for increased cell yields revealed increases in some subpopulations.

Potentiation of murine cytomegalovirus pneumonitis by antibiotics in clinical use.

Nineteen antibiotics were screened for their effects on the proliferation of murine spleen cells in vitro. Ketoconazole suppressed lymphoproliferation at clinically-attainable concentrations, whilst tetracycline, cephalothin, rifampicin and ciprofloxacin were also inhibitory at relatively low concentrations. These antibiotics were selected for further study. High concentrations of cephalothin inhibited macrophage uptake of colloidal gold, while spleen cells from mice treated with ketoconazole responded poorly to mitogenic stimulation in vitro. Humoral responses to ovalbumin, polyvinylpyrrolidone and murine cytomegalovirus (MCMV) were not suppressed by oral administration of ketoconazole, tetracycline, cephalothin, rifampicin or ciprofloxacin to mice. However, MCMV-infected mice receiving these antibiotics had increased virus loads and a greater persistence of virus and interstitial pneumonitis in their lungs. This was observed with clinically-attainable serum concentrations of cephalothin, tetracycline and ciprofloxacin. The findings warrant further investigation as the antibiotics are used to control secondary infections in immunosuppressed patients, many of whom experience cytomegalovirus disease.