Marianne Hokland

The effect of interferon on lymphocyte-mediated effector cell functions: Selective enhancement of natural killer cells

Abstract The effect of human interferons on different types of lymphocyte-mediated killer assays was explored. Killing by T cells generated through mixed lymphocyte cultures as well as antibody-dependent lymphocyte-mediated cytotoxicity was not influenced by the addition of interferon. Enhancement of cytolysis produced by natural killer cells was observed when interferon was added during the assay, but enhancement could also be induced if the effector cells were pretreated with interferon for 2 hr prior to the lytic reaction. Killing of a cell line susceptible to natural killing was increased and a cell line which is normally relatively resistant to this type of killing became a susceptible target.

Secreted and Membrane-Associated Matrix Metalloproteinases of IL-2-Activated NK Cells and Their Inhibitors

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.

Is there an association between rumination and self-reported physical health? A one-year follow-up in a young and an elderly sample.

Cross-sectional studies have suggested an association between rumination and subjective health. The aim of the present study was to investigate in a longitudinal design whether rumination was related to self-reported physical health. A total of 96 young (age range 20–35) and 110 elderly (age range 70–85) participants completed questionnaires measuring rumination, negative affect, life events, and self-reported physical health at baseline and at 1-year follow-up. Multiple linear regressions showed a significant association between self-reported physical health at time 1 only for the elderly and negative affect mediated the association. At follow-up, rumination was significantly associated with self-reported physical health only for the young and the association was only partly mediated by negative affect. In conclusion, rumination is associated with poorer self-reported physical health, but the association depends on the age of the individual as well as time span studied.

Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer

In this article we report about the role that tumor structure and extracellular matrix (ECM) may play in immunotherapy and in gene therapy using adenoviruses. We performed studies in a rat model for colorectal cancer, CC531, and in specimens of human colorectal cancer. The tumors were composed of two compartments, tumor cell nests surrounded by stromal cells. ECM proteins were expressed in the stromal part, where the blood vessels were also located. Furthermore, in several tumors, the tumor cell nests were surrounded by basal membrane-like structures. Therefore, in vascular approaches to treat cancer, therapeutic agents on their route to tumor cells may be hampered by ECM to reach tumor cells. We found that immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumor cells in the CC531 rat model. Tumor cells were only infected, and even then in limited numbers, in cases of intratumoral injection. We hypothesize that ECM in a tumor is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and thus limits colorectal tumor therapy.

Monocytes and neutrophils as ‘bad guys’ for the outcome of interleukin-2 with and without histamine in metastatic renal cell carcinoma – results from a randomised phase II trial

Histamine (HDC) inhibits formation and release of phagocyte-derived reactive oxygen species, and thereby protects natural killer (NK) and T cells against oxidative damage. Thus, the addition of histamine may potentially improve the efficacy of interleukin-2 (IL-2). We have explored this potential mechanism clinically in two randomised phase II trials in metastatic renal cell carcinoma (mRCC). In parallel with the clinical trial in Denmark (n=63), we obtained serial blood samples and tumour biopsies searching for a potential histamine effect in situ. At baseline and on-treatment weeks 3 and 8, we monitored the ‘good guys’ (i.e. NK and T cells) and ‘bad guys’ (i.e. monocytes/macrophages and neutrophils) simultaneously in blood (n=59) and tumour tissue (n=44). Patients with high number of monocytes and neutrophils in peripheral blood had very poor survival, with apparently no benefit from either IL-2 alone or IL-2/HDC treatment. Blood monocytes (r=−0.36, P=0.01) and neutrophils (r=−0.46, P=0.001) were negatively correlated with cytotoxicity, whereas blood NK cells were positively correlated with cytotoxicity (r=0.39, P=0.002). Treatment with IL-2 alone resulted in a significantly higher number of circulating monocytes (P=0.037) and intratumoral macrophages (P=0.005) compared with baseline. In contrast, IL-2/HDC resulted in an unchanged number of circulating monocytes and intratumoral macrophages, and in addition, a significantly increased number of intratumoral CD56+ NK cells (P=0.008) and CD8+ T cells (P=0.019) compared with baseline. The study provides evidence that circulating monocytes and neutrophils are powerful negative prognostic factors for IL-2-based immunotherapy and establishes a biological rationale for the potential use of histamine in conjunction with IL-2 in mRCC.

Tumor‐localization by adoptively transferred, interleukin‐2‐activated NK cells leads to destruction of well‐established lung metastases

We have shown previously that i.v. injection of interleukin‐2‐(IL‐2) activated natural killer (A‐NK) cells together with IL‐2 leads to a substantial localization of the A‐NK cells into most, but not all, well‐established B16 lung metastases in C57BL/6 mice within 12–24 hr. We demonstrate that the morphology of the lung metastases, (loose or more compact in appearance), and their location in the lungs (on the surface or deep in the lung parenchyma) are closely tied to the infiltration‐permissiveness of the metastases as well as their sensitivity to treatment with A‐NK cells. Although more than 1,100 A‐NK cells/mm2 were found in deep metastases with a “loose” morphology (D‐L), only 534, 90 and 89 cells/mm2 were found in surface‐loose (S‐L), surface‐compact (S‐C) and deep‐compact (D‐C) metastases, respectively. The best infiltrated metastases responded best to the A‐NK cell therapy. Thus, metastases of the D‐L phenotype became reduced by 65–90% after treatment with 2 × 106 A‐NK cells and IL‐2 (120,000 IU Peg‐IL‐2 every 12 hr for 3 days) compared to similar lesions in animals treated with PEG‐IL‐2 alone. In contrast, poorly infiltrated metastases, that is lesions of the compact phenotype (D‐C and S‐C) as well as loose metastases on the lung surface (S‐L), did not react significantly to this treatment. We conclude that adoptively transferred A‐NK cells are able to eliminate even well‐established metastases. The existence of metastases that are resistant to infiltration by the transferred effector cells at time of treatment might reduce the efficacy of cell‐based immuno‐therapeutic ventures.

EFFECT OF PSYCHOLOGICAL INTERVENTION IN THE FORM OF RELAXATION AND GUIDED IMAGERY ON CELLULAR IMMUNE FUNCTION IN NORMAL HEALTHY SUBJECTS : AN OVERVIEW

The present study measured the effects of relaxation and guided imagery on cellular immune function. During a period of 10 days 10 healthy subjects were given one 1-hour relaxation procedure and one combined relaxation and guided imagery procedure, instructing the subjects to imagine their immune system becoming very effective. Even though no major changes in the composition of the major mononuclear leukocyte subsets could be demonstrated a significant increase in natural killer function was demonstrated. These data suggest that relaxation and guided imagery might have a beneficial effect on the immune defense and could form the basis of further studies on psychological intervention and immunological status.

Antitumor activity of NK cells

NK cells have been shown to play an important role in the lungs with regards to tumor cell clearance and resistance of this organ to metastases. Here, we have investigated whether NK cells play a similar role in organs other than the lungs. We conclude that while organ-resistance to metastases correlates well with the NK activity of the host, a clear correlation between NK activity and clearance of tumor cells is found only in the lungs. We also demonstrate that activation of NK cells with the TLR 3 ligand poly I:C results in a substantial increase in the number of organ-associated NK cells. This increase may explain the increased resistance to metastasis seen in many organs after poly I:C treatment. Finally, we present data showing that NK cells activated ex vivo with IL-2 are able to localize to lung tumors following iv adoptive transfer and to significantly reduce the tumors they infiltrate. We conclude that NK cells, which currently are under intense investigation owing to their newly discovered immunoregulatory functions, remain very potent antitumor killer cells capable of killing not only circulating tumor cells, but also well-established micro metastases.

Structural Insight into the Function of Myelin Basic Protein as a Ligand for Integrin αMβ2

Multiple sclerosis (MS) is an inflammatory disease where phagocytic cells infiltrate the nerve tissue and act as terminal agents in destruction of the myelin sheath. However, the mechanism that triggers the ability of these cells to recognize myelin remains obscure. We show that myelin basic protein (MBP), a major autoantigen in MS, is a potent and specific ligand for the integrin αMβ2 (Mac-1, CD11b/CD18) expressed mainly on phagocytic cells. MBP undergoes a dramatic conformational change when liberated from the lipid-rich environment of the myelin sheath. The MS drug glatiramer acetate mimics the conformationally labile regions of MBP, interacts in the unfolded state strongly with αMβ2, and inhibits the MBP binding to αMβ2. Our study reveals a link between MBP, glatiramer acetate, and the αMβ2 integrin, and suggests a new model for MS pathogenesis based on the recognition of unfolded MBP by the αMβ2 integrin.

Intratumoural and peripheral blood lymphocyte subsets in patients with metastatic renal cell carcinoma undergoing interleukin-2 based immunotherapy: association to objective response and survival.

The aim of the present study was to analyse lymphocyte subsets in consecutive peripheral blood samples and consecutive tumour tissue core needle biopsies performed before and during interleukin-2 based immunotherapy, and to correlate the findings with objective response and survival. Twenty-six patients with metastatic renal cell carcinoma were treated with low dose s.c. interleukin-2, interferon-α and histamine. A total of 250 blood samples and 62 core needle biopsies from 23 and 19 of these patients, respectively, were analysed. After 2 weeks of treatment, a significant positive correlation between absolute number of peripheral blood lymphocytes (P=0.028), CD3 (P=0.017), CD57 (P=0.041) and objective response was demonstrated. There was no correlation between any peripheral blood leukocyte subsets and survival. Cytotoxicity of peripheral blood mononuclear cells was not correlated to objective response or survival. Within the tumour tissue at baseline, a significant positive correlation between CD4 (P=0.027), CD8 (P=0.028), CD57 (P=0.007) and objective response was demonstrated. After one month of immunotherapy, a significant positive correlation between intratumoral CD3 (P=0.026), CD8 (P=0.015), CD57 (P=0.009) and objective response was demonstrated. A significant positive correlation between intratumoral baseline CD4 (P=0.047), baseline CD57 (P=0.035), CD3 at one month (P=0.049) and survival was demonstrated. These data provide novel in vivo evidence of the possible contribution of lymphocyte subsets in the tumour reduction in responding patients during interleukin-2 based immunotherapy. Confirmation of the results requires further studies including a larger number of patients.