Immune Modulating Effects of Lentinan

The antitumor activity of lentinan resulted from activation of the host's immune functions rather than direct cytotoxicity to target cells (22,26,66,67, 69,70). The mechanism is postulated to involve binding of h-glucan to the surface layer of lymphocyte or specific serum protein. This activated macrophages, T cells, NK cells, and other effector cells, as well as increasing production of antibodies, interleukins, and interferons (45,71,72). Lentinan is considered to be phagocytosed by cells of macrophage lineage present in organs such as liver, spleen, and lung, and activates these cells (54,55,70,7375). Macrophages are the first to recognize foreign bodies as nonself and give this information to lymphocytes to activate the immune system. They probably respond acutely to BRMs such as lentinan. Lentinan has been shown to facilitate the infiltration of lymphocytes and macrophages into tumor tissues (64).

Suzuki et al. (55-57) also showed that the antitumor effect was dependent on the CD-positive T lymphocytes. Lentinan stimulates the macrophages to augment their antitumor activity (76). More macrophages and T cells were found in lentinan-fed mice compared to the control group. Suzuki and co-workers (55) and Hamuro et al. (57) suggested that in addition to the augmentation of immune effector cell activity against tumors, infiltration of these cells into the tumor sites might also be involved in eradication of tumors by lentinan. Tumor-induced immunosuppression could also be overcome by lentinan treatment through enhancement of the macrophage migration inhibitory factor production (32).

The effector cells might act either selectively or nonselectively on target cells (8). Various kinds of bioactive serum factors, associated with immunity and inflammation, (such as IL-2, IL-3, vascular dilation inducer, and acute-phase protein inducer), appeared immediately after the administration of lentinan. Increments in the production of antibodies as well as interleukins (IL-1, IL-2) and interferon (IFN-g) have also been observed (12,13,22,26,57, 63,67,70,77-86). The mechanism of lentinan-enhanced antibody-dependent, cell-mediated cytotoxicity through helper T cells (77,84) remains uncertain.

Yap and Ng further conducted a time-sequenced study on the induction of various cytokines in AKR mouse after oral feeding with lentinan. Four cytokines levels, namely IL-1a (interleukin-1 produced mainly by monocytes, promote proliferation of Th2 CD4+ T cells), IL-2 (interleukin-2 produced mainly by Th0 and Th1 CD4+ cells, as T-cell growth factor), TNF-a (produced mainly by monocytes, activates endothelial cells and other cells of immune and nonimmune systems), and IFN-g (interferon-gamma, produced by Th1 CD4+ cells, activates NK cells, macrophages, and killer cells), rose significantly after feeding with lentinan. They peaked at different hours after feeding with lentinan but returned to baseline after 24 hr (Fig. 3a-d). The IL-1a (Fig. 3a) and IL-2 (Fig. 3b) peaked at 2 hr postfeeding with lentinan. Both the TNF-a and IFN-g peaked at 4 hr postfeeding (Fig. 3c and 3d).

These results suggested that oral administration of lentinan may serve as a means of activating the immune system, provoking the immune responses required for disease prevention. Lentinan, once ingested, may encounter the gut-associated lymphoid tissue (GALT), which is a well-developed immune network, evolved to protect the host from infecting pathogens. Lentinan may also be absorbed into the systemic circulation, thereafter, involved in inducing immune systems against future pathogenic attack. Quantitative analysis of orally administered lentinan in murine blood carried out using limulus colorimetric test demonstrated that pure lentinan was detected in the murine blood and peaked at 0.2 mg (equivalent to the usual intravenous or intraperitoneal inoculation dosage) 30 min after feeding (Yap and Ng, unpublished data).

When fed with crude mushroom homogenates, IL-1a, IL-2, and TNF-a levels were also induced but to much lower levels when compared to lentinan-fed cohort. The IFN-g level was not significantly induced. The lipid and protein fractions from the mushrooms did not result in noticeable induction of any of the four cytokines tested and was at the baseline like the buffer-fed cohort (control).

Many interesting biological activities of lentinan have been reported. This included an increase in the activation of nonspecific inflammatory responses such as APP (acute-phase protein) production (87), vascular dilation, and hemorrhagic necrosis of the tumors (88). Bradykinin-induced skin reaction could be used as an index of vascular reactions against lentinan and this skin reaction could be used to moniter the host sensitivity to lentinan in antitumor responses (89).

Activation and generation of helper and cytotoxic T cells (10,67,79,9093) is an essential aspect of lentinan treatment. The augmentation of immune mediators such as IL-1 and IL-3, colony-stimulating factor(s) (94), migration inhibitory factor (32,94,95), and increasing the capacity of PBM (peripheral blood mononuclear) cells (52,53) contributed additively to the antitumor efficacy. Wang and Lin (96) postulated that the immunomodulating effect of lentinan might be relevant to change of T-cell subpopulation and increase of TNF production.

The recent study of Yap and Ng confirmed that T-lymphocytes were increased significantly (fourfold, p < 0.001, Student's t-test) after feeding with lentinan (Table 2). The CD3, CD4 (T-helper), and CD8 (T-cytotoxic) lymphocytes were isolated using T-cell-enrichment columns. All three types of CD lymphocytes were activated in comparison with the buffer-fed controls. It was noted that the placebo (controls) effects did result in some degree of

Figure 3 Cytokine profiles from mice fed with lentinan, crude mushroom homogenate, lipid fraction, protein fraction, and buffer (placebo). (a) IL1-a profile. There is a sharp rise 2 hr after feeding with lentinan. A small increase in level of IL1-a is seen in the mice fed with the crude mushroom homogenate. For the other three cohorts of mice, the level is at the baseline. (b) IL-2. Similar to (a), a significant rise in IL-2 level is seen in the lentinan-fed mice. This is followed in modest amount in the crude-mushroom-homogenate-fed population. Again the lipid-, protein-, and buffer-fed mice do not show any change in the level over the experimental period. (c) TNF-a. The rise in TNF-a level is seen at 4 hr after lentinan feeding. The level is also quite significant for the crude-mushroom-homogenate-fed mice. The usual low level of stimulation is seen with the other three regimes. (d) IFN-g. The profile obtained is similar to (c) with rise at 4 hr in the lentinan-fed mice. Other than a low level of induction seen in the crude-mushroom-homogenate-fed population, the other three cohorts show minimal changes in the level of production over the 24-hr period.

Figure 3 Cytokine profiles from mice fed with lentinan, crude mushroom homogenate, lipid fraction, protein fraction, and buffer (placebo). (a) IL1-a profile. There is a sharp rise 2 hr after feeding with lentinan. A small increase in level of IL1-a is seen in the mice fed with the crude mushroom homogenate. For the other three cohorts of mice, the level is at the baseline. (b) IL-2. Similar to (a), a significant rise in IL-2 level is seen in the lentinan-fed mice. This is followed in modest amount in the crude-mushroom-homogenate-fed population. Again the lipid-, protein-, and buffer-fed mice do not show any change in the level over the experimental period. (c) TNF-a. The rise in TNF-a level is seen at 4 hr after lentinan feeding. The level is also quite significant for the crude-mushroom-homogenate-fed mice. The usual low level of stimulation is seen with the other three regimes. (d) IFN-g. The profile obtained is similar to (c) with rise at 4 hr in the lentinan-fed mice. Other than a low level of induction seen in the crude-mushroom-homogenate-fed population, the other three cohorts show minimal changes in the level of production over the 24-hr period.

activations of the lymphocytes. This could be due to the response of stress from force feeding of the fluid. The most activated lymphocytes were the CD4 helper lymphocytes (100%) followed by almost similar degree of activation in CD3 and CD8 lymphocytes.

Lentinan-activated lymphocytes were extracted from lentinan-fed AKR mice and reinoculated into nude (athymic) and SCID (lack immuno-globulin and lymphocytes) mice before the inoculation of human colon carcinoma cells. Six different cell lines were used representing different stages of differentiation of the carcinoma cells. Tabulated in Figure 4, the data showed that the "lentinan-activated'' lymphocytes did indeed protect the immunocompromised mice effectively against human colon carcinoma development (49). These experiments showed beyond doubt that T-lym-phocytes were truly activated by lentinan administration. The antitumor mechanism was a T-cell-mediated process. Figure 5 shows the proposed immunomodulating pathway after lentinan administration. Activation of

TABLE 2 Cluster of Differentiation of T-Lymphocytes

Mice fed with

Total cell count (CD90— normal)

Cell count (CD25 — activated)

Percentage of activation (%)

CD3

Lentinan

13.55 x

106

9.405

x

106

69.20

T-lymphocytes

Crude

3.989 x

106

6.200

x

106

64.24

isolated using

mushroom

CD3 T-cell-

homogenate

enrichment

Buffer solution

2.313 x

106

4.593

x

106

50.69

column

CD4 T helper

Lentinan

2.150 x

106

2.150

x

106

100.00

lymphocytes

Crude

1.435 x

106

0.715

x

106

49.85

isolated using

mushroom

CD4 T-cell-

homogenate

enrichment

Buffer solution

0.782 x

106

0.311

x

106

40.33

column

CD8 T

Lentinan

3.393 x

106

2.263

x

106

66.80

cytotoxic

Crude

2.148 x

106

0.875

x

106

39.29

lymphocytes

mushroom

isolated using

homogenate

106

106

CD8 T-cell-

Buffer solution

1.515 x

0.523

x

34.54

enrichment

column

the immune responses against the tumor cells resulted in the process of apoptosis in these cells leading to tumor regression (49).

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