Kazuko Uno

Augmenting effect of opioid peptides on murine macrophage activation

We investigated the effect of several opioid peptides on the activation of murine peritoneal exudate macrophages (M phi) in vitro. M phi were treated with interferon (IFN) as a priming agent and bacterial lipopolysaccharide (LPS) as a triggering agent in the presence or absence of opioid peptides. M phi activation was assessed by their tumoricidal activity. When treatment with IFN and LPS resulted in a high level activation of M phi, dynorphin-A exerted no further enhancing effect. When treatment induced only weak activation, however, dynorphin-A augmented the M phi activation. Leucine-enkephalin, methionine-enkephalin, and also beta-endorphin had augmenting effects. An opioid receptor antagonist, naloxone, reduced the effect of dynorphin-A and beta-endorphin. When M phi were treated sequentially with IFN and LPS, beta-endorphin operated in combination with LPS only. Moreover, beta-endorphin was effective for already activated M phi. These results indicate that opioid peptides act on M phi via classical opioid receptors, and that responsiveness to opioid peptides is induced in the triggering stage of M phi activation.

Suppressive effect of recombinant TNF-gelatin conjugate on murine tumour growth in-vivo

Abstract— A recombinant human tumour necrosis factor α (TNF) was conjugated to gelatin by means of carbodiimide to improve the in‐vivo stability of TNF. About 55% of TNF activity was retained after gelatin conjugation as judged by the cytotoxicity assay using L‐M cells in‐vitro. Intraperitoneal injection of the TNF‐gelatin conjugate significantly suppressed the in‐vivo growth of murine Meth A fibrosarcoma cells (SS2 cells) in mouse peritoneum, in comparison with that of free TNF at the same dosage (P < 0·05). After intraperitoneal injection, TNF activity of the conjugate was detected in both the serum and the peritoneal cavity of mice for a longer period than was free TNF, irrespective of the presence of SS2 cells. Chromatographic studies of the conjugate demonstrated that the increase in the apparent molecular weight of TNF was consistent with gelatin conjugation. It is likely that this leads to a prolonged retention of TNF activity in‐vivo. In addition, the TNF‐gelatin conjugate suppressed the in‐vitro growth of SS2 cells to the same extent as free TNF. Thus, it is possible that the longer retention period of the conjugate brought about an increase in the chance of contact between TNF and SS2 cells, resulting in the enhanced suppressive effect of TNF on in‐vivo tumour cell growth. Gelatin conjugation is an effective method for increasing the in‐vivo antitumour activity of TNF.

In vivo Effects of Recombinant Interferon Alpha A/D Incorporated in Gelatin Microspheres on Murine Tumor Cell Growth

Intraperitoneal (ip) injections of gelatin microspheres containing a very small amount of recombinant human interferon alpha A/D (A/D‐IFN) (IFN‐microspheres) plus free A/D‐IFN improved the survival of mice bearing ascitic Meth A‐R1 cells which we had isolated as IFN‐resistant cells under in vitro conditions. The dose of free A/D‐IFN in one injection was 10,000 IU, which was insufficient by itself for manifesting in vivo antitumor activity. In these mice, in vivo Rl cell growth was suppressed and macrophage recruitment was enhanced in comparison with mice receiving other control agents. Administration of IFN‐microspheres alone was also effective but less than that of IFN‐microspheres plus free A/D‐IFN. Peritoneal macrophages obtained from normal or R1‐bearing mice receiving ip injection of IFN‐microspheres with or without free A/D‐IFN were activated to inhibit the in vitro growth of R1 cells. The intratumoral injection of IFN‐microspheres strongly inhibited the growth of solid R1 tumors. Intravenous injection of IFN‐microspheres was effective in preventing the pulmonary metastasis of B16 melanoma cells. These results indicate that the IFN‐microsphere is much more effective against tumors than free A/D‐IFN.

Murine endothelial cell line cells, F-2: interaction with leukocytes and cytokines production.

Flowcytometry demonstrated that murine endothelial cell line F‐2 expresses MHC class I antigen, FcR II, Mac‐1 and vascular cell adhesion molecule‐1 (VCAM‐1), but not intercellular adhesion molecule‐1 (ICAM‐1) and class II antigen. However, co‐culturing with TNF‐α for 24 hr resulted in the increased expression of ICAM‐1, and the decreased expression of VCAM‐1. IL‐1α and IFN‐γ exerted this regulatory effect on VCAM‐1 but not on ICAM‐1. T (Con A blast) and B (LPS blast) cells adhered to F‐2 cells at almost equal levels, and the adhesion was enhanced 20 to 50% when the cells were precultured with TNF‐α for 24 hr. The inhibition assay using either (anti‐ICAM‐1 + anti‐LFA‐1, lymphocyte function‐associated antigen‐1) or (anti‐VCAM‐1 + anti‐VLA‐4, very late antigen‐4) mAbs demonstrated that the ICAM‐1 system was utilized more preferentially by T than B blasts when F‐2 cells were stimulated with TNF‐α, and the VCAM‐1 system was vice versa under the unstimulated and stimulated conditions. Granulocytes also adhered to F‐2 cells, but no mAbs could inhibit the adhesion. Although F‐2 cells produced a considerable amount of IL‐6, GM‐CSF and neutrophil chemotactic activity, a 24 hr incubation with TNF‐α resulted in an increase of 12 fold in IL‐6 and 3 fold in neutrophil chemotactic activity production.

Alteration of Physiological Activity of Activated Macrophages through L‐Arginine Metabolism

Our aim in this study was to define the effect of L‐arginine on macrophages (Mø) in relation to the decay of tumoricidal activity of activated Mø. We found that the activated M0 retained their cytotoxicity when cultured in L‐arginine‐deficient medium but not in conventional medium. Such a decline of tumoricidal activity was associated with increase of glucose consumption and concomitant lactate production, resulting in Mø death. Addition of glucose to the culture medium of activated Mø appeared to cause only a slight delay of the decrease of tumoricidal activity and Mø death. These events were also coincident with a decrease of electron transport activity in mitochondria. Cytological observation by electron microscopy clearly showed the structural alteration or destruction of mitochondria, which preceded the changes of other physiological and functional activities. These results demonstrate that the L‐arginine‐dependent cytolytic activity against tumor target cells also impairs M0 functions and ultimately induces Mø death, which is primarily mediated by the inhibition of mitochondrial activity.

Potentiation of in vivo Antitumor Effects of Recombinant Interleukin‐1α by Gelatin Conjugation

Chemical conjugation of a recombinant human interleukin‐1α (IL‐1) with gelatin was conducted using a water‐soluble carbodiimide in an attempt to augment the indirect effect of IL‐1 on in vivo tumor cell growth in mice. Chromatographic studies of the IL‐1‐gelatin conjugate demonstrated that the apparent molecular weight of IL‐1 was increased by the gelatin conjugation and about 60% of IL‐1 activity was retained in the prepared conjugate. Intraperitoneal (i.p.) injection of the conjugate significantly suppressed the intraperitoneal growth of a subline of Meth A fibrosarcoma cells (RR1 cells), compared with the effect of free IL‐1 at the same dose, although the cells per se were resistant not only to free IL‐1 but also to gelatin‐conjugated IL‐1. Simple mixing of gelatin with free IL‐1 did not augment the in vivo antitumor effect as compared with that of free IL‐1. Gelatin conjugation improved the in vivo stability of IL‐1. Prolonged retention of IL‐1 activity in the peritoneal cavity as well as the circulation of mice was observed after i.p. injection of the IL‐1‐gelatin conjugate in comparison with free IL‐1 injection, irrespective of the presence of tumor cells. Gelatin conjugation was effective in augmenting the in vivo antitumor effects of IL‐1 to activate host cells, e.g. macrophages (Mø). The i.p. injection of the conjugate enhanced Mø infiltration into the peritoneal cavity of tumor‐bearing mice and peritoneal Mø were strongly activated to inhibit the in vitro growth of RR1 cells. Thus, gelatin conjugation was effective in augmenting the indirect effect of IL‐1 via host cells, leading to a high suppressive effect on in vivo growth of tumor cells.

Susceptibility to Infection Due to Diminished Interferon-α-Producing Capacity in Patients with Refractory Anemia with Excess of Blasts or Refractory Anemia with Excess of Blasts in Transformation

Peripheral whole blood cells from normal subjects and from 8 patients with refractory anemia with excess of blasts (RAEB) and one patient with RAEB in transformation (RAEB-t) were studied to ascertain their interferon (IFN)-α-producing capacity. This capacity was determined by time-resolved immuno-fluoroassay 20 hrs after the blood cells had been incubated with 500 HA/ml of Sendai virus. The mean IFN-α-producing capacity in the peripheral whole blood cells of 35 normal males was 8718 ± 4683 IU/ml, while that of 49 normal females was 7549 ± 4731 IU/ml; not a significant difference. All of the RAEB and RAEB-t patients examined showed a significantly low (<2500 IU/ml) IFN-α-producing capacity in the peripheral whole blood cells. In addition, the levels of IFN-α-producing capacity in 1 ml cell suspension containing 1 x 10(6) peripheral mononuclear blood cells were markedly lower in all 5 RAEB patients and the RAEB-t patient examined than those in the normal subjects, when 5 HA of Sendai virus was used. The activities of natural killers were lower in the RAEB/RAEB-t patients than those in the normal subjects. These findings suggest that IFN-α-producing capacity is diminished in the peripheral whole blood cells of RAEB and RAEB-t patients, and great care is advised regarding infection by virus and bacteria in treatment of these patients.