Cytokine regulation of constitutive production of interleukin-8 and -6 by human pancreatic cancer cell lines and serum cytokine concentrations in patients with pancreatic cancer.

Patients with pancreatic cancer frequently demonstrate symptoms such as weight-loss and muscle wasting and have clinical evidence of a systemic inflammatory response. Such effects may be mediated by pro-inflammatory cytokines derived from tumor cells. The production of interleukin-6 and -8 by pancreatic cancer cell lines and the influence of other cytokines on this production was studied. IL-8 was produced by all cell lines and production was increased following exposure to IL-1 and TNF. Cytokine-stimulated, but not basal IL-8 production was reduced by co-incubation with IL-4 in the MIA PaCa-2 and PANC-1 cell lines. The CFPAC cell line produced IL-6, but this production was not altered by IL-1, TNF or IL-4. In the PANC-1 cell line IL-8 and IL-8 receptors were only detected by PCR in cells which had been stimulated with TNF or IL-1. Serum concentrations of IL-6 and IL-8 were elevated in patients with pancreatic cancer compared with controls. In conclusion, human pancreatic cancer cell lines elaborate pro-inflammatory cytokines which have the potential to mediate elements of the systemic inflammatory response.

Effect of interleukin-2 on peripheral blood mononuclear cell cytokine production and the hepatic acute phase protein response.

This study investigated the ability of recombinant interleukin-2 (IL-2) to modulate the ability of peripheral blood mononuclear cells (PBMCs) to stimulate an acute phase protein response in isolated human hepatocytes. The effect of IL-2 on the production of tumour necrosis factor-alpha (TNF) and interleukin-6 (IL-6) by PBMCs isolated from patients with gastrointestinal cancer, multiple organ failure, and healthy controls was also studied. The ability of supernatants from IL-2-treated PBMCs to elicit an acute phase response in hepatocytes was then investigated. IL-2 had no effect on IL-6 or TNF production by PBMCs isolated from any group in the presence or absence of bacterial lipopolysaccharide (LPS). Despite this, preincubation of PBMCs with IL-2 significantly reduced the potential of LPS-stimulated PBMC supernatants to stimulate production of alpha1 antichymotrypsin, alpha1-acid glycoprotein, and C-reactive protein by hepatocytes. These observations were not due to a direct effect of IL-2 on hepatocyte acute phase protein production. These findings suggest that in this model IL-2 may modulate PBMC-induced acute phase protein production through an IL-6 and TNF-independent pathway.

Endogenous production of IL-8 by human colorectal cancer cells and its regulation by cytokines.

This study investigated the endogenous production of the pro-inflammatory and angiogenic cytokine IL-8 by human colorectal cancer cells. We describe substantial (>0.5 ng/ml) constitutive production of IL-8 by certain human colorectal cancer cell lines without the requirement for exogenous stimulation. Tumour cell-derived IL-8 production was upregulated by the addition of the pro-inflammatory type 1 cytokines TNF alpha, IL-1 beta and IFN gamma. Addition of the regulatory type 2 cytokines IL-4 and IL-10 produced paradoxical stimulation of IL-8 production in certain cell lines and downregulation of IL-8 production in others. These results suggest that tumour-derived cytokine production may have an important role in patients with colorectal cancer. Furthermore, they demonstrate the complexity of tumour cell cytokine production and highlight the difficulties in developing effective therapeutic biological response strategies.

Effect of interleukin-4 on pro-inflammatory cytokine production and the acute phase response in healthy individuals and in patients with cancer or multiple organ failure.

1. This study investigates whether previously documented effects of interleukin-4 in down-regulating pro-inflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) from healthy individuals would be reproducible in PBMCs isolated from patients with multiple organ failure (acute disease model) and gastrointestinal cancer (chronic disease model). The effects of interleukin-4 on the ability of PBMC supernatants to elicit an acute phase protein response from isolated human hepatocytes were also studied. 2. Incubation of PBMCs with interleukin-4 significantly reduced both spontaneous and lipopolysaccharide-induced production of tumour necrosis factor and lipopolysaccharide-induced interleukin-6 production, demonstrating that the PBMCs from patients with acute and chronic disease are not refractory to the effects of interleukin-4. The effects of interleukin-4 on the ability of PBMCs from the groups studied to elicit an acute phase response were complex and varied both between patient groups and individual acute phase proteins. Overall, interleukin-4 reduced the potential of PBMCs to stimulate production of the positive acute phase proteins C-reactive protein, alpha1-antichymotrypsin and alpha1-acid glycoprotein.3. This work emphasizes the pleiotropic nature of cytokines and the complex regulatory mechanisms which exist. The study illustrates the difficulties in devising in vivo intervention strategies using cytokines such as interleukin-4.

Interleukin-8 can mediate acute-phase protein production by isolated human hepatocytes.

During the course of studies designed to identify the role of cytokines in the reprioritization of hepatic protein synthesis associated with cachexia we detected a hepatocyte-stimulating moiety in the supernatants of pancreatic cancer cells that was unrelated to interleukin (IL)-6. This study identifies that moiety as IL-8 and investigates the role of IL-8 in the induction of acute-phase protein production. The human pancreatic cancer cell line MIA PaCa-2 produced >1 ng/ml of IL-8 per 24 h, and supernatants from this cell line induced C-reactive protein (CRP) production from isolated human hepatocytes. Addition of neutralizing anti-human IL-8 antibody to such supernatants produced almost complete inhibition of CRP production. The addition of recombinant human IL-8 to hepatocytes resulted in a dose-dependent increase in CRP, alpha1-acid glycoprotein, and alpha1-antichymotrypsin production and a decrease in the production of transferrin and prealbumin. This study demonstrates that recombinant or tumor-derived IL-8 can modulate acute-phase protein production from isolated human hepatocytes and from human hepatoma cells.

Down-regulation of the acute-phase response in patients with pancreatic cancer cachexia receiving oral eicosapentaenoic acid is mediated via suppression of interleukin-6.

1. Weight loss in pancreatic cancer is associated with persistent elevation of the acute-phase protein response. The effect of oral administration of eicosapentaenoic acid on the regulation of the acute-phase response in weight-losing patients with pancreatic cancer was investigated in vitro and in vivo. 2. Oral supplementation with eicosapentaenoic acid, in patients with cancer cachexia, resulted in a significant reduction in the serum concentration of the acute-phase protein C-reactive protein (11.0 +/- 4.8 mg/l before eicosapentaenoic acid compared with 0.8 +/- 0.8 mg/l after 4 weeks of eicosapentaenoic acid, P < 0.05), but no significant reduction in the serum concentration of the hepatocyte-stimulating cytokine interleukin-6. Production of interleukin-6 by peripheral blood mononuclear cells isolated from patients was significantly reduced after supplementation with eicosapentaenoic acid (interleukin-6 production by peripheral blood mononuclear cells exposed to 10 micrograms of lipopolysaccharide/ml: 10.2 +/- 2.1 ng/ml before supplementation with eicosapentaenoic acid compared with 3.5 +/- 1.7 ng/ml after supplementation, P < 0.05) and supernatants from these cells had reduced potential to stimulate C-reactive protein production by isolated human hepatocytes (hepatocyte C-reactive protein production in response to supernatants from peripheral blood mononuclear cell cultures exposed to 10 micrograms of lipopolysaccharide/ml: 150.4 +/- 18.6 ng/ml before eicosapentaenoic acid versus 118 +/- 14.9 ng/ml after 4 weeks of eicosapentaenoic acid, P < 0.05). The potential of lipopolysaccharide-stimulated peripheral blood mononuclear cell supernatants to stimulate C-reactive protein production by hepatocytes could be attenuated by neutralizing anti-interleukin-6 antibody in control subjects and in patients before, but not after, treatment with eicosapentaenoic acid. 3. In conclusion, eicosapentaenoic acid can down-regulate the acute-phase response in patients with pancreatic cancer cachexia and this process is likely to involve suppression of interleukin-6 production.

Proinflammatory cytokine release by peripheral blood mononuclear cells from patients with acute pancreatitis.

Proinflammatory cytokine release was measured from peripheral blood mononuclear cells (PBMCs) isolated from six volunteers and, on admission, from 16 patients with acute pancreatitis. Tumour necrosis factor (TNF) release in patients did not differ significantly from that of volunteers, whereas both interleukin (IL) 6 and IL-8 release in patients was raised when compared with that in the volunteer group (mean(s.e.m.) IL-6 20.7(4.6) versus 9.3(1.7) ng/ml, P = 0.03; IL-8 283(40) versus 128(22) ng/ml, P = 0.04). When variation in white cell count was accounted for, IL-6 and IL-8 release but not that of TNF was significantly greater in patients with severe disease than in those with mild disease. These results point to a complex upregulation of proinflammatory cytokine release from PBMCs in patients with acute pancreatitis, components of which relate to the clinical progress of the disease.

Ibuprofen reduces energy expenditure and acute-phase protein production compared with placebo in pancreatic cancer patients.

The aim of this study was to investigate the effect of the cyclo-oxygenase inhibitor ibuprofen on the acute-phase protein response and resting energy expenditure (REE) of weight-losing patients with pancreatic cancer. Patients with irresectable pancreatic cancer (n = 16) were treated with either ibuprofen (1200 mg day-1 for 7 days (n = 10) or placebo (n = 6). A group of 17 age-related non-cancer subjects were also studied. Indirect calorimetry, anthropometry, multifrequency bioelectrical impedence analysis and serum C-reactive protein (CRP) estimation were performed immediately before and after treatment. Before treatment, total REE was significantly elevated in the pancreatic cancer patients compared with healthy controls (1499 +/- 71 vs 1377 +/- 58 kcal) (P < 0.02). Following treatment the mean REE of the ibuprofen group fell significantly (1386 +/- 89 kcal) compared with pretreatment values (1468 +/- 99 kcal) (P < 0.02), whereas no change was observed in the placebo group. Serum CRP concentration was also reduced in the ibuprofen-treated group (pre-ibuprofen, 51 mg l-1; post-ibuprofen, 29 mg l-1; P < 0.05). These results suggest that ibuprofen may have a role in abrogating the catabolic processes which contribute to weight loss in patients with pancreatic cancer.

Interleukin-4 and interleukin-10 increase endotoxin-stimulated human umbilical vein endothelial cell interleukin-8 release.

The aim of this study was to determine the effect of interleukin-4 (IL-4) and interleukin-10 (IL-10) on interleukin-8 (IL-8) release from endothelial cells. Confluent monolayers of human umbilical vein endothelial cells (HUVECs) were incubated in the absence or presence of 10 ng/ml of bacterial lipopolysaccharide (LPS), with 5% human AB serum and recombinant human IL-4 or IL-10 over a dose range from 50 fg/ml to 50 ng/ml (final concentration). IL-4 and IL-10 had no effect on HUVEC IL-8 release in the absence of LPS. In the presence of LPS, IL-4 and IL-10 enhanced IL-8 release by approximately 300% compared with LPS-stimulated cells alone, IL-8 release increasing from 2594 +/- 493 pg/ml (no IL-4 or IL-10) to 7892 +/- 320 pg/ml (IL-4, 5 pg/ml; p = 0.001) and 8359 +/- 712 pg/ml (IL-10, 50 pg/ml; p = 0.002). IL-8 release in response to IL-4 or IL-10 plateaued above 5 and 50 pg/ml, respectively. This study suggests that IL-4 and IL-10 may be involved in the complex regulation of endothelial cell cytokine production during the response to endotoxin.

Murine HIV-1 p24 specific T lymphocyte activation by different antigen presenting cells: B lymphocytes from immunized mice present core protein to T cells.

Mice were injected with three doses of baculovirus-produced recombinant HIV-1 p24 core protein in alum adjuvant. CD4 positive T lymphocytes from immunized animals proliferated in vitro in the presence of antigen and peritoneal macrophages (Mps) or splenic dendritic cells (DCs) from non-immunized mice as antigen presenting cells (APCs). DCs were approximately three times more efficient than Mps on a cell for cell basis. No synergy was observed between Mps and DCs in this system. B lymphocytes from immunized animals also presented p24 antigen to the specific T cells. Mps did synergize with B cells to enhance the level of T lymphocyte proliferation. This may have implications for the induction of specific immune responses to pathogens after administration of single protein vaccines.

Antigen presentation in patients with recrudescent orofacial herpes simplex virus infections.

Recovery from epidermal herpes simplex virus (HSV) infection depends primarily on development of an effective cell-mediated immune response, possibly generated following antigen (Ag) presentation by epidermal cells (EC). The ability of EC to present HSV Ag was investigated in 12 subjects with occasional recrudescent facial HSV infections. All had circulating HSV specific antibodies and cell-mediated immunity to the virus. Peripheral blood mononuclear cell suspensions, depleted of antigen presenting cells (APC) by glass adherence and then enriched for T cells by adsorption on nylon wool columns, did not proliferate in response to HSV Ag. Both EC suspensions, prepared from suction blister roofs, and glass-adherent peripheral blood mononuclear cells (AC) preincubated with ultraviolet-inactivated HSV, reconstituted the T-cell proliferative response to HSV. EC were more efficient than AC at presenting HSV Ag to T cells. Depletion of CD1+ cells from EC suspensions by cell sorting reduced their ability to present HSV Ag and augmentation of CD1+ cell numbers supplemented it. Preincubation of EC or AC with monoclonal antibodies to major histocompatibility complex class II antigens DP, DQ or DR, blocked the lymphoproliferative response to HSV Ag. Evidence was obtained that cells co-ordinately expressing products of the DP, DQ and DR loci are involved in presentation of HSV Ag by both EC and AC.

Severe eczema herpeticum is associated with prolonged depression of cell-mediated immunity to herpes simplex virus.

Nine patients with eczema herpeticum (EH) and 2 with atopic eczema and recurrent cold sores were investigated for cell-mediated immunity (CMI) and antibodies (Ab) to herpes simplex virus (HSV). All patients had Ab to HSV and a high lymphoproliferation response to concanavalin A and all but 3 showed lymphoproliferation to HSV antigens. These 3 patients had very severe EH and absent CMI to HSV for many months after clinical recovery despite normal Ab and concanavalin A responses. In 2 of them, depletion of CD8+ T cells by panning restored the lymphoproliferative response to HSV. Thus their absent CMI may be due to suppressor cell function rather than lack of responsive cells.

Lymphoproliferative and serological responses to herpes simplex virus during primary infection, recrudescence, and re-infection in a murine model.

Mice were infected epidermally with herpes simplex virus type 1 (HSV-1) after mild tape stripping. Some were re-infected by the same route several weeks later; recrudescences were induced in others by UV-irradiation before the primary infection followed by re-irradiation and tape stripping at a later date. Clinical symptoms were noted; serological responses to HSV and lymphoproliferative and phenotypic analysis of local lymph node cells were measured throughout. Experience of a primary lesion did not prevent lesions developing again on re-infection although morbidity and mortality were decreased. Recrudescent lesions were less severe than primary or secondary lesions, never zosteriform and healed rapidly. Antibodies to HSV were not found to play a major role in preventing development of lesions. The lymphoproliferative response on primary infection was maximal just after the lesions were most severe and then waned. There was a second, although not accelerated, lymphoproliferative response on re-infection with a persisting high level for at least one month. On recrudescence, limiting dilution culture analysis of lymphoproliferation demonstrated a recruitment within two days of HSV-1 specific lymphocytes to lymph nodes draining sites of lesions, which may limit their severity and duration.

The immune response to glycoproteins of herpes simplex virus.

Herpes simplex virus commonly causes 'cold sores', the virus often becoming latent after the primary infection and recrudescing at intervals. Six glycoproteins have been described in the virion and in the membranes of infected cells; much of the host's immune response is directed against these molecules. The functions of individual glycoproteins in the disease process and in the induction of different components of the immune response are beginning to be unravelled, which should help provide a strategy for the creation of an effective subunit vaccine against the virus.

Immune responses to herpes simplex virus in patients with facial herpes simplex and those with eczema herpeticum.

The immune response to herpes simplex virus (HSV) was studied in 59 patients with primary and recrudescent facial HSV infections. The patients included nine with atopic eczema, seven of whom had eczema herpeticum (EH). All patients had antibodies to HSV (measured by ELISA) and all but three had HSV-specific cell mediated immunity (CMI) (measured by in vitro lymphoproliferation). Thirteen control subjects were negative for both tests. All three patients with absent CMI to HSV had suffered from severe EH and had depressed CMI to HSV for several months following an attack. In two of these EH patients, a positive CMI response was produced by in vitro removal of CD8 + ve T lymphocytes from peripheral blood mononuclear cells using a panning technique. Thus the absence of CMI to HSV in these patients was due to suppressor cell function rather than a lack of specifically responsive cells. The other four EH patients with normal CMI to HSV had suffered less severe EH, but no association between the absence of CMI to HSV and serum IgE level or activity of the eczema was apparent in the atopic patients. No specific anti-HSV IgE antibody was detectable.

The role of antigen presentation in the ontogeny of immune responses to herpes simplex virus.

The ability of epidermal antigen presenting cells (APC) to induce immune responses to herpes simplex virus (HSV) has been studied in mice of differing ages. Using an in vivo model of HSV infection we have demonstrated that neonatal epidermal cells (EC) induce specific suppression of DH to HSV in normal syngeneic adult mice. The suppression is transferable and mediated by T cells of the Lyt1+, Lyt2-, L3T4+ phenotype. The ability of EC to induce suppression persists up to 4 weeks of age, whereas EC from mice 6 weeks of age or older induce positive DH responses to the virus. This correlates with the susceptibility of mice of the different ages to HSV infection and their ability to mount DH responses to the virus.

A murine model of herpes simplex virus recrudescence.

A murine model is described in which recrudescence of herpes simplex virus (HSV) type 1 was achieved. C3H mice were shaved and irradiated with u.v. B light 3 days before being infected epidermally with a clinical isolate of HSV. Seven weeks or longer following the primary infection, the survivors were again shaved, irradiated with u.v. and mildly tape-stripped. Recrudescent lesions occurred in up to 80% of mice at the site of the original lesion in most cases, but also occasionally at other sites. Skin painting with u.v.-irradiated urocanic acid (a substance suggested to be a photomediator of the immunosuppressive effects of u.v.) in place of u.v.-irradiation induced some recrudescence but was not as efficient as u.v.-irradiation. Antibody titres to HSV had no value in predicting whether recrudescence would occur but lymphoproliferative responses in draining lymph nodes may provide some indication of viral activity at the epidermal site. A hypothesis is developed that u.v.-irradiation before primary infection with HSV induces a suppressive immune response to the virus which affects the virus-host interaction and accounts for a high incidence of recrudescent lesions on subsequent stimulus.

In vivo modulation of antigen presentation generates Ts rather than TDH in HSV-1 infection.

The role of suppressor cells in control of persistent infections may be of profound importance. Whether a positive immune response or suppression of immunity is generated at the time of initial exposure to pathogens causing such infections may in part be due to the nature of the initial antigen presentation to the specific cells of the immune system. We have shown that in vivo modulation of epidermal APCs by an environmentally encountered stimulus (UV-B light exposure) and subsequent transfer of these APCs together with live HSV-1 to naive syngeneic recipients is sufficient to generate suppression of DH rather than DH to HSV-1. This suppression is T-cell mediated and specific for HSV-1. The phenotype of the Ts cell induced by epidermal cell transfer is Thy1+ L3T4+ Ly2-.

Induction of suppression of delayed type hypersensitivity to herpes simplex virus by epidermal cells exposed to UV-irradiated urocanic acid in vivo.

Urocanic acid (UCA), the putative photoreceptor for ultraviolet radiation (UV)-induced suppression, undergoes a UV-dependent trans to cis isomerisation. Epidermal cells from mice painted with UCA, containing a known proportion of the cis-isomer, generate suppression of the delayed type hypersensitivity response to herpes simplex virus type 1 (HSV-1) when transferred to naive syngeneic recipients at the same time and site as infection with HSV-1. One T suppressor cell subset, of phenotype (Thy1+, L3T4+, Ly2-), is induced by the cis-UCA modified epidermal cell transfer. Flow cytometric analysis of the epidermal cells from skin treated with UV or cis-UCA indicates an overall reduction from normal in the number of cells expressing MHC Class II antigens, but no alteration in the number expressing I-J antigens.

Alterations in epidermal handling of HSV-1 antigens in vitro induced by in vivo exposure to UV-B light.

In order to investigate the cellular interactions involved in the immune response to herpes simplex virus type 1 (HSV-1) in a murine model, an in vitro antibody induction system was developed. This comprised HSV-1-primed T cells from infected mice, trinitrophenol (TNP)-primed B cells from mice primed with TNP-coupled calf erythrocytes, and TNP-HSV-1 as antigen. When antigen-presenting cells (APC) were removed from the assay system, the induced antibody response disappeared but could be reconstituted by the addition of APC derived from the peritoneal cavity or skin of normal mice. Since HSV-1 is an epidermal pathogen, it was decided to investigate the role of skin APC in HSV-1 immunity. Skin APC from mice irradiated 3 days previously with a suberythemal dose of ultraviolet (UV)-B were found to have a decreased capacity to present HSV-1 antigen in vitro. However, the APC capacity of their peritoneal cells was unaffected. The reduction in APC capacity is not only a local effect at the irradiated site, as APC from mice exposed to UV-B with one ear protected by black electrical tape were equally affected in both ears.