Perioperative immunotherapy with recombinant interleukin 2 in patients undergoing surgery for colorectal cancer.

Major surgery impairs the cellular immune response. We have therefore studied the immunological effects of low-dose recombinant interleukin 2 given to patients undergoing surgery for colorectal cancer to determine whether this agent has potential in perioperative adjuvant immunotherapy. Patients were randomly allocated to control (n = 13) or treatment groups (n = 12). Immunological studies of both lymphocyte function and subset number were performed preoperatively and on Days 1, 4, 7, and 10. Treatment with recombinant interleukin 2 prevented the postoperative fall in both natural killer and lymphokine-activated killer cell cytotoxicity, clearly demonstrated in the control group. The treatment group also showed in vivo T-cell activation with an initial lymphopenia followed by a rebound lymphocytosis and upregulation of the subset markers CD25 (interleukin 2 receptor) and CD45RO (T-memory cells). These combined effects may have important consequences in controlling metastatic dissemination of tumor during the vulnerable perioperative period.

The response to interferon of NK and K cells from conventionally immunosuppressed and cyclosporine-treated renal allograft recipients.

Interferon is a potent stimulator of natural killer (NK) and killer (K) cell activity in human beings, both these cytotoxic functions representing host defense mechanisms against viral infections and lymphoid malignancy. Both NK and K cell functions are markedly impaired in conventionally immunosuppressed allograft recipients but coincubation of lymphocytes from these patients with purified human lymphoblastoid interferon considerably augments both these activities. Cyclosporine immunosuppression causes only a moderate, but significant, impairment of NK activity--but K cell activity appears to be normal. Again IFN increases NK activity of the lymphocytes of these patients but produces a fall or only moderate increases in K cell activity. We conclude that these data support the functional distinction between NK and K cells and suggest that immunosuppressive agents act at the pre-NK/K cell stage of maturation, though possibly via different mechanisms.

Interleukin-10. A role in the development of postoperative immunosuppression.

BACKGROUND: The cause of diminished monocyte major histocompatibility complex class II antigen expression after surgery or trauma is unclear. Interleukin-10 (IL-10) regulates inflammatory cytokine production and major histocompatibility complex class II (HLA-DR) expression in vitro. OBJECTIVES: To quantify in vivo IL-10 messenger RNA (mRNA) and protein and monocyte HLA-DR expression after major surgery and to investigate the effects of IL-10 neutralizing blockade on monocyte HLA-DR expression in vitro. DESIGN: Inception cohort study of 48 surgical patients from preoperative status to postoperative day 7 and 9 healthy volunteers (controls). SETTING: Large teaching hospital, Northern England. PATIENTS: Monocyte HLA-DR and cytokine mRNA expression was determined in 32 of 48 consecutive patients undergoing elective major resectional surgery. Mononuclear cells for in vitro studies and serum samples for IL-10 measurement were obtained from the remaining 16 patients. MAIN OUTCOME MEASURES: Monocyte HLA-DR expression determined by flow cytometry, IL-10, and tumor necrosis factor mRNA in peripheral blood mononuclear cells assayed by multiplex reverse transcriptase polymerase chain reaction, and serum IL-10 determined by enzyme-linked immunosorbent assay. RESULTS: Monocyte HLA-DR expression (in mean channel fluorescence units [MCF]) was significantly reduced 24 hours after surgery (MCF [+/- SEM], 32.6 +/- 2.3 vs 16.3 +/- 1.2; P < .001) and remained low during the first postoperative week. A relative increase in IL-10 to G3PDH mRNA ratio (mean [+/- SEM], 0.95 +/- 0.08 vs 0.59 +/- 0.06; P < .01) and serum IL-10 (mean [+/- SEM], 18.1 +/- 4.1 vs 5.4 +/- 0.8 pg/mL; P < .01) was noted on the first postoperative day. A significant correlation existed between HLA-DR antigen expression and the presence of IL-10 mRNA transcript on the first postoperative day (P < .01). Lipopolysaccharide-induced up-regulation of monocyte HLA-DR expression was significantly impaired on the first postoperative day (mean [+/- SEM], 151% +/- 24.4% vs 60% +/- 10.1%; P < .01), but this was partially reversed by IL-10 neutralizing antibody (mean [+/- SEM], 60% +/- 10.1% vs 115% +/- 11.6%; P < .01). CONCLUSIONS: Interleukin-10 gene expression correlates with the fall in monocyte HLA-DR antigen expression in patients undergoing major abdominal surgery and may account for the immunosuppression associated with surgical injury.

Does oxpentifylline ('Trental') have a place in the treatment of intermittent claudication?

In a randomized double-blind study, the clinical and haemorrheological responses of 40 patients receiving oxpentifylline (200 mg 3-times daily) were compared with those of 40 patients receiving placebo. The treatment period in both groups was 2 months. The parameters measured before and after treatment were: subjective response; claudication and maximum walking distances; ankle systolic indices; maximum blood flow in the lower limb by gravimetric plethysmography; plasma fibrinogen; erythrocyte deformability and whole blood viscosity. There was a significant increase (p less than 0.05) in mean erythrocyte deformability in the oxpentifylline group but not in the placebo group; this apparent difference between the groups, however, was not significant. The placebo group showed a significant improvement (p less than 0.05) in claudication distance and mean plasma fibrinogen concentration, but no such improvements were observed in the oxpentifylline group. There were no significant differences in either of the two groups with regard to the subjective response, ankle systolic indices, maximum limb blood flow or whole blood viscosity. It is concluded that oxpentifylline , when taken in oral form at the dose used in this study, increased erythrocyte deformability without conferring any clinical or haemorrheological benefit to patients with intermittent claudication.

Total parenteral nutrition adversely influences tumour-directed cellular cytotoxic responses in patients with gastrointestinal cancer.

The effects of total parenteral nutrition (TPN), using a lipid-based regimen, on non-specific lymphocyte function and tumour-directed cellular cytotoxicity was studied in 30 patients suffering from gastrointestinal (GI) cancer. After 7 days of TPN, augmented lymphocyte blastogenesis and production of the helper T-lymphocyte lymphokine Interleukin-2 were observed. However, over the same time period, significant impairment of basal natural killer (NK), and IL-2 activated NK activity were observed. Furthermore, lymphokine-activated killer (LAK) cell responses towards the NK resistant cell line DAUDI and the colorectal tumour cell line COLO 320, were markedly depressed. These findings have important implications for the use of this TPN regimen in GI cancer patients who might be considered for either surgical adjuvant or primary treatment with immunotherapy protocols.

Sequential changes in NK and ADCC effector cell function after organ transplantation in man.

Antibody-dependent cellular (K cell) cytotoxicity (ADCC) and cellular cytotoxicity against natural killer (NK) targets exerted by the same cells were both measured sequentially in ten patients after renal transplantation using conventional immunosuppression. Cytotoxicity against NK targets appeared to correlate more closely with rejection episodes than did assays of ADCC activity during the same period. An apparent rise in NK activity was frequently accompanied by a fall in ADCC activity. Collectively these data suggest that the previously reported increase in NK activity during rejection episodes may represent a manifestation of the appearance of non-clonally activated T-cells in peripheral blood during rejection.

Pharmacological concentrations of lipid emulsions inhibit interleukin-2-dependent lymphocyte responses in vitro.

Most immunological functions are accomplished by means of interactions between mediator molecules (cytokines or lymphokines) and their specific receptors on the lymphocyte surface. One particular lymphokine, Interleukin-2 (IL-2) is central to the generation of most immune responses including those with antitumor activity. Prompted by two clinical trials which have suggested distinct but apparently opposite effects of lipid emulsions on the production of and lymphocyte responses to IL-2 we have examined the effects of pharmacological concentrations of three lipid emulsions currently in clinical use on IL-2 related interactions in vitro. Mitogen-stimulated and IL-2 activated human lymphocyte proliferation were both inhibited in a dose-dependent manner in the presence of all three lipid emulsions although the effects were less marked with the solution in which 50% of the calories are present as medium-chain triglycerides (MCT) rather than long-chain triglycerides (LCT). Similarly the LCT, but less so the MCT-containing solutions inhibited the generation of cytotoxic lymphokine-activated killer cells. These solutions did not inhibit the proliferation of cell lines which are not growth-factor dependent but did inhibit the growth of an IL-2-dependent cell line. We conclude that lipid emulsions can upset IL-2-dependent lymphocyte responses. These observations may lead to parenteral feeding regimens which are less immunocompromising for the tumor-bearing patient.

Autologous transfusion: an alternative to transfusion with banked blood during surgery for cancer.

Autologous blood transfusion in surgery for cancer has been avoided because of the metastatic potential of reinfused malignant cells. This study determined whether viable tumour cells remain in the red cell concentrate after separation and whether blood transfusion filters remove these tumour cells before reinfusion. Units of banked blood were inoculated with tumour cell lines: breast cancer SKBr3; colon cancer COLO 320; lymphoma Daudi; erythroleukaemia K562. After processing with the Cell Saver, aliquots of the red cell concentrate and waste saline wash were examined for tumour cells and cultured. Tumour cells from all four cell lines were identified in the red cell concentrate but not in the waste saline wash. All the cell lines except Daudi grew from the red cell concentrate. Experiments on two of the cell lines (SKBr3 and COLO 320) were performed in which the red cell concentrate was either unfiltered (control) or filtered with SQ40S blood transfusion filter or RC100 leucocyte depletion filter. Both cell lines were present in the control samples and after filtration with SQ40S filters, and cells from these samples grew normally in culture. No tumour cells were evident after filtration with the RC100 filters and no growth of either cell line was found after 1 week in culture. The Cell Saver in combination with RC100 filters may be suitable for use during the surgical treatment of malignant disease.

Natural killer-cell activity, interferon-alpha 2 production, and interleukin-2 production in cyclosporine-treated and conventionally immunosuppressed human allograft recipients.

Natural killer (NK) activity, interferon (IFN)-alpha production, and interleukin-2 (IL-2) production were measured in renal transplant recipients undergoing immunosuppression with either azathioprine and steroids (Az + P) or cyclosporine (CyA). Overall, both IFN-alpha production and IL-2 production were impaired in these two groups compared with identical studies in healthy individuals. However, on the basis of control data these two patient groups were divided into those with "normal" NK activity and those with "low" NK activity. In the CyA group those with a low NK reaction produced less IL-2 and IFN-alpha than those with normal NK activity. No such relationship between cytokine production and NK activity was discerned in the Az + P group. These data conflict with in vitro studies, which have failed to demonstrate any effect of CyA on IFN-alpha production. In addition, they suggest that whereas cyclosporine influences NK activity in vivo by inhibiting cytokine production, other factors may play a role in impairing NK activity in conventionally immunosuppressed patients.

Inhibition of lymphokine-activated killer cell generation by cultured tumor cell lines in vitro.

The co-culture of human peripheral blood mononuclear cells (PBMC) with high concentrations of interleukin 2 normally generates lymphokine-activated killer (LAK) cells capable of indiscriminate lysis of tumor targets. However, the addition of certain cell-line-derived tumor cells to the LAK generation cultures within the first 48 h of culture initiation resulted in the suppression of the LAK cytotoxicity measured after 3-4 days of culture. Suppression could be achieved with tumor cell:PBMC ratios as low as 1:50 when tumor cells were derived from melanoma and colorectal cancer (G361, COLO320, HT-29), but suppression was not observed with cells from the breast cancer cell line SKBr3. No suppression of LAK generation was observed with normal epithelial cells from colon or breast, with autologous or allogeneic lymphoblasts, or with allogeneic vascular endothelial cells. Suppression was independent of the removal of adherent cells from PBMC, could not be prevented by indomethacin and was not attributable to interleukin 2 absorption/adsorption by tumor cells. The suppressive activity of some tumor cells could be augmented by preculture in recombinant gamma interferon. Serum-free supernatants from G361, COLO320 and HT-29 (but not SKBr3 or endothelial cells) were also highly suppressive towards the generation of LAK cells. The elaboration by tumor cells of factors capable of inhibiting LAK generation may partially explain the failure of LAK/interleukin 2 therapy in some experimental and clinical protocols.

The effect of 5-fluorouracil and alpha interferon and 5-fluorouracil and leucovorin on cellular anti-tumour immune responses in patients with advanced colorectal cancer.

Interferon alpha (IFN-alpha) enhances the activity of 5-fluorouracil (5-FU) in the treatment of advanced colorectal cancer although the mechanism is not understood. We have investigated the effect of this combination on cellular immunity and compared this with standard therapy of 5-FU/L-leucovorin, in 24 patients with advanced colorectal cancer. This study has demonstrated an enhancement of the cellular immune response in patients given 5-FU/IFN-alpha with augmentation of natural killer (NK) cell function and abrogation of 5-FU-induced suppression of lymphokine-activated killer (LAK) cell activity.

Immunorestorative effect of lipid emulsions during total parenteral nutrition.

This prospective in vivo cross-over study investigated the effect of Intralipid on immune responses. Twenty-three patients were randomly allocated to receive one of two alternative total parenteral nutrition (TPN) regimens for the first 7 days and the other regimen for the second 7 days. Only one of the regimens included a fat emulsion to provide 50 per cent of the calorific requirement. Immunological studies were performed on days 0, 7 and 14. These included peripheral blood T cell subsets, antibody dependent cellular cytotoxicity and basal and maximal Interleukin 2 production. All immunological parameters were significantly augmented during total parenteral nutrition using the lipid based regimen. No such change was seen during intravenous feeding with carbohydrate based TPN. It is concluded that, far from being immunosuppressive, the incorporation of a fat emulsion into a TPN regimen has immunostimulatory properties.

Enhanced polymorphonuclear leucocyte adhesion after surgical injury.

OBJECTIVE: To evaluate the relationship between the expression of the neutrophil adhesion receptor CD11b, functional neutrophil adhesion, and clinical outcomes in patients undergoing major surgical resections. DESIGN: Laboratory study. SETTING: University hospital, United Kingdom. SUBJECTS: 25 patients undergoing surgical resections for malignant disease. INTERVENTIONS: Blood obtained preoperatively and on postoperative days 1, 3, and 6. Neutrophils were assayed for CD11b expression in whole blood and following isolation using flow cytometry, and adhesion was measured using a chromogenic, gelatin-dependent adhesion assay, with and without stimulation by FMLP. Postoperative outcomes were classified as uncomplicated (n = 20) or sepsis according to ACCP/SCCM definitions (n = 5). RESULTS: Whole blood neutrophil CD11b expression was significantly increased on the first postoperative day and in all patients was significantly higher in the sepsis group (p < 0.001, ANOVA). There was a significant increase in CD11b expression above preoperative concentrations after separation and stimulation with FMLP only in the sepsis group, 3 and 6 days postoperatively (p < 0.01, paired t-test). Functional neutrophil adhesion was significantly higher in the sepsis compared with the uncomplicated group at all time points postoperatively (p < 0.001, two way ANOVA). CONCLUSION: Major operations are accompanied by upregulation of baseline CD11b expression mirrored by changes in neutrophil adhesion. Those patients who develop sepsis have greater degrees of CD11b expression and adhesion. These data present a potential pathophysiological role for neutrophils throughout the postoperative period in the development of sepsis and its sequelae after major operations.

Suppression of the generation of lymphokine-activated killer (LAK) cells by serum-free supernatants of in vitro maintained tumour cell lines.

Serum-free supernatants from in vitro maintained gastrointestinal cancer and melanoma cell lines inhibit the generation of lymphokine (IL-2) activated killer (LAK) cells in a time and dose-related manner. Concentrations as low as 5% can inhibit the generation of LAK cytotoxicity but inhibition of proliferation is not observed until higher concentrations are included in the culture system. Inhibition is not observed with supernatants from a breast cancer cell line nor with supernatants from normal cells. There was complete concordance between the capacity of the tumour cells themselves to inhibit LAK generation and the presence of inhibitory activity in the corresponding supernatant. The inhibitory factor(s) is stable after heating to 44 and 56 degrees C. Production of the inhibitory factor(s) is sensitive to metabolic inhibitors and has a molecular weight greater than 25 kD. The inhibition of LAK cell stimulation by tumour cells may partially explain the failure of adoptively transferred LAK cells and IL-2 therapy to cause tumour regression in man.

Peri-operative modulation of cellular immunity in patients with colorectal cancer.

The peri-operative cellular immune response is depressed in patients with gastrointestinal cancer, a factor which may facilitate malignant dissemination. We have investigated the effects of peri-operative rIL-2 and a combination of rIL-2 and interferon-alpha (IFN-alpha) on both peripheral blood lymphocyte function and number in patients undergoing surgical resection for colorectal cancer. Fifty-two patients were randomly allocated to either control, rIL-2 or rIL-2 with IFN-alpha treatment arms. In vitro studies were performed pre-operatively and on post-operative days 1, 4, 7 and 10. Natural killer (NK) and lymphokine-activated killer (LAK) cell function were profoundly depressed in control patients (P < 0.001; P < 0.01), an effect abrogated in both treatment groups; indeed NK function was augmented in the rIL-2 and IFN-alpha group on the first post-operative day in association with an increase in the percentage of cells expressing CD16 and CD56 (P < 0.01). Flow cytometric analysis of lymphocyte subsets in the control group was unremarkable, except for an early post-operative fall in numbers of lymphocytes. Treatment with either rIL-2 or rIL-2 and IFN-alpha produced an initial profound reduction in T lymphocyte numbers, followed by a 'rebound' lymphocytosis of activated CD3+ T cells, as demonstrated by a significant increase in co-expression of CD25, CD38 and CD45RO. No significant differences were observed between either of the treatment groups. Adjuvant immunotherapy affects peri-operative anti-tumour immune responses, and this may influence long term outcome in patients undergoing surgery for gastrointestinal cancer.

Monocyte activation after open and laparoscopic surgery.

BACKGROUND: The level of expression of major histocompatibility complex class II antigens, human leucocyte antigen (HLA)-DR, on monocytes correlates with the development of sepsis after surgery or trauma. Normally interferon (IFN) gamma can increase monocyte HLA-DR expression and thus may be a potential biological prophylactic antisepsis agent in patients suffering from accidental or surgical trauma. The purpose of this study was to determine the capacity of monocytes to respond to IFN-gamma after conventional open surgery and laparoscopic surgery of similar magnitude. METHODS: A whole blood flow cytometric assay was used to measure monocyte HLA-DR antigen expression before and after monocyte activation with either IFN-gamma or bacterial lipopolysaccharide (LPS). Blood was obtained before operation and on postoperative day 1 from 43 patients undergoing conventional open surgery and from 15 undergoing laparoscopic surgery of similar magnitude. RESULTS: Whole blood monocyte HLA-DR expression fell significantly after both conventional open and laparoscopic surgery. IFN-gamma caused monocyte HLA-DR expression to rise by a median (interquartile range) of 43 (26-60) per cent and 63 (10-124) per cent before operation in the open and laparoscopic groups respectively. However, after operation the corresponding values were 20 (6-41) per cent and 26 (9-74) per cent (P < 0.003 in all cases). Identical findings but of greater magnitude were observed with LPS stimulation before and after operation in the two surgical groups. CONCLUSION: Monocyte HLA-DR expression is diminished by surgical operations and is relatively refractory to further stimulation by IFN-gamma or LPS after surgery. Laparoscopic surgery is as suppressive as conventional surgery in this regard. The resistance of postoperative monocytes to further activation by IFN-gamma suggests that this agent may be ineffective as a biological response modifier after major surgery or trauma.

Effects of different lipid emulsions on lymphocyte function during total parenteral nutrition.

A prospective randomized study of the immunological effects of three total parenteral nutrition (TPN) regimens in patients undergoing preoperative parenteral nutrition was conducted. In one regimen the calories were derived solely from glucose. The others were identical except that 50 per cent of the calories were provided as lipid emulsion, in one as long-chain triglycerides (LCT) only while the other contained half the fat as medium-chain triglycerides (MCT) and half as LCT (MCT/LCT). Natural killer (NK) activity and lymphokine-activated killer (LAK) activity were significantly higher after TPN with the MCT/LCT solution. A significant fall in LAK activity occurred after TPN with the LCT solution. The interleukin 2 content in supernatants from activated T lymphocytes was significantly higher after TPN with the LCT-containing solution. Solutions containing LCT and those containing MCT perturb cytokine interactions, but this is less with MCT-containing solutions, which may augment certain responses. These observations may have implications for the design of TPN regimens.

Lymphokine activated killer (LAK) cells in patients with gastrointestinal cancer.

Lymphokine activated killer (LAK) cells are a recently described cellular immune phenomenon with exciting potential for the treatment of tumours arising from solid organs. A comparison of some aspects of LAK cell precursors and LAK cell function was undertaken in 44 control subjects and 44 preoperative patients suffering from gastrointestinal cancer (20 localised and 24 advanced). Lymphokine activated killer cell precursor (natural killer (NK) cell) activity was significantly diminished in patients with advanced tumours (p less than 0.02) as was fully mature LAK cell activity against an NK resistant target cell (p less than 0.012). T-lymphocyte responses were not significantly different between the three groups. The reduced LAK cell generation was associated with a significantly diminished proliferative response of LAK precursors to stimulation with high dose IL-2 in vitro (p less than 0.012). Impaired LAK cell generation may explain the failure of adoptive cellular immunotherapy with LAK cells in some patients with advanced gastrointestinal cancer and prompts the search for means of augmenting this activity in such patients.

Augmentation of lymphokine-activated killer cell activity in patients with gastrointestinal cancer.

Adoptive cellular immunotherapy with lymphokine-(interleukin 2) activated killer (LAK) cells is not as successful in patients with gastrointestinal cancer as with other tumour types. This may be because the cytotoxic capacity of LAK cells from such patients is suboptimal. In this study we have sought to augment this activity by stimulating the lymphocytes with recombinant human interferon-gamma (r-HuIFN-gamma) in addition to interleukin 2 or by depleting the lymphocytes of adherent suppressive mononuclear cells. Both procedures augment LAK activity in gastrointestinal cancer patients but adherent cell depletion results in fewer cells being available for adoptive cellular immunotherapy. No further augmentation of LAK activity of adherent cell depleted cells could be accomplished by addition of r-HuIFN-gamma. Co-stimulation of unfractionated peripheral lymphocytes with r-HuIFN-gamma is the preferable procedure for the generation of LAK cells for adoptive cellular immunotherapy in patients suffering from gastrointestinal cancer.

Effects of low dose perioperative interferon on the surgically induced suppression of antitumour immune responses.

General surgical procedures are followed by a period of generalized immunosuppression that may favour the deposition of metastases seeded at operation in patients with malignant disease. In an attempt to prevent the suppression of host-antitumour immune mechanisms following surgery we have studied the immunological effects of low-dose perioperative interferon-alpha (r-HuIFN alpha). Patients were randomly allocated pre-operatively to the control (n = 15) or treatment group (n = 15). Patients in the treatment arm received a 1-week course of subcutaneous recombinant human interferon-alpha 2a (Roferon-A) at a dose of 2 megaunits daily starting on the evening before surgery. Natural killer cell, lymphokine activated killer cell cytotoxicities and endogenous interleukin 2 production were measured 1 day before surgery and on the first, third, fifth and tenth postoperative days. Treatment with r-HuIFN alpha did not prevent the postoperative impairment of interleukin 2 production or lymphokine activated killer cell cytotoxicity. However it prevented the fall in natural killer cell activity normally observed following surgery. This may have important consequences in controlling metastatic dissemination of tumour in this vulnerable period.