CD137 Ligand Activities on Monocytes and Macrophages

CD137 ligand is expressed constitutively on peripheral monocytes and mono-cyte/macrophage cell lines (Table 3.2).

The signal through CD137 ligand activates monocytes and the CD137 lig-and signal alone is sufficient for activation. When CD137 ligand is crosslinked by recombinant CD137 protein or an anti-CD137 ligand antibody, it induces adherence of monocytes within a few hours. The adherent monocytes change their shape over the course of a week and adopt the three basic macrophage morphologies, which are round, elongated, and branched cells (Langstein and Schwarz, 1999). Concomitantly with adherence the CD137 ligand signaling induces the expression of proinflammatory cytokines (TNF, IL-6, IL-8, IL-12) and activation markers (ICAM-1), and it inhibits expression of anti-inflammatory cytokines (IL-10) and differentiation markers (FcyRIII), (Laderach et al., 2003; Langstein et al., 1998).

Table 3.1. Activities of CD137 Ligand Signaling by Cell Type. (Trg.: CD137 Hgand-transgenic; Def.: CD137 ligand-deficient; H: human; M: mouse; S: species.)

Cell type

S

Activity

System

Reference

Monocytes

H

Induction of TNF, IL-6, IL-8,

in vitro

Langstein et al., 1998;

Macrophages

IL-12, ICAM and

Langstein and Schwarz,

adherence. Induction of

1999; Langstein et al.,

M-CSF and prolongation

1999; Langstein et al.,

of survival. Induction of

2000; Laderach et al.,

proliferation and

2003; Ju etal., 2003

endomitosis. Inhibition of

FcyRIII and IL-10

M

Amplification of cell numbers

Trg. mice

Zhu etal., 2001

Dendritic cells

H

Induction of IL-12, CD11c,

in vitro

Kim etal., 2002;

CD86, CD137 ligand,

Laderach et al. , 2003

MHC class II and cellular

adherence

M

Induction of IL-6, IL-12,

in vitro

Futagawa et al. , 2002

CD80 and CD86

B cells

H

Costimulation of

in vitro

Pauly et al., 2002

proliferation and

immunoglobulin secretion

M

Costimulation ofproliferation

in vitro

Pollok etal., 1994

M

Elimination of peripheral B

Trg. mice

Zhu etal., 2001

cells

T cells

H

Inhibition of proliferation

in vitro

Schwarz et al., 1996; Michel

and induction of cell death

etal., 1999; Ju etal., 2003

by apoptosis

M

Inhibition of proliferation

Def. mice

Kwon et al. , 2002

Bone marrow

M

Induction of proliferation.

in vitro

Saito et al., 2004

cells

Inhibition of osteoclast

differentiation

Carcinoma cells

H

Induction of IL-8

in vitro

Salih et al., 2000

The degree of monocyte activation by CD137 ligand signaling is dose-dependent. IL-8 release correlates with the concentration of recombinant CD137 protein (Langstein et al., 1998) and reducing the CD137 signal strength also delays adherence and morphological changes (unpublished observation).

The process of monocytes adherence is at least in vitro associated with a cellular activation. However, activation induced by CD137 ligand signaling is not merely a consequence of the monocytes starting to adhere, since activation can also be induced through CD137 ligand when the cells are cultured under non-adherent conditions. However, crosslinking of CD137 ligand is essential for activation as soluble CD137 protein is inactive and has no effects on monocytes (Langstein etal., 1998).

The CD137 ligand signal also induces expression of macrophage colony-stimulating factor (M-CSF), an essential survival factor for monocytes and significantly prolongs survival of monocytes in vitro (Langstein and Schwarz, 1999a). Neutralizing anti-M-CSF antibodies block CD137 ligand-mediated cell survival

Table 3.2. CD137 Ligand Protein Expression by Tissue. Listed are Only Reports Which Demonstrate CD137 Ligand Expression at the Protein Level. (Const.: constitutive; H: human; M: Mouse; S: species.)

Cell type

S

Expression

Reference

T cells

- Primary, resting

H

Negative

Salih et al., 2000

- Jurkat, CEM

H

Constitutive

Palma et al., 2004

- T cells clones

H

Inducible by anti-CD3

Alderson etal., 1994

- Primary

M

Negative

Futagawa et al. , 2002

- Primary

M

Const., low levels, not inducible

Pollok etal., 1994

- T cell clones

M

Const., low levels, not inducible

Pollok etal., 1994

- EL-4, WR-19L, BW5147

M

Constitutive

Futagawa et al. , 2002

-L5178Y

M

Negative

Futagawa et al. , 2002

B cells

- Primary

H

Induced by pock weed mitogen

Zhou etal., 1995

- Primary

H

Constitutive

Jung etal., 2004

- Daudi, SKW6.4

H

Constitutive

Zhou etal., 1995

- Raji, IM-9

H

Constitutive

Palma et al., 2004

- Primary

M

Induced by CD40L and cAMP

DeBenedette etal., 1997

- 2PK-3, A20

M

Constitutive

Pollok etal., 1994

- K46J, M12

M

Constitutive, enhanced by

DeBenedette etal., 1995

cAMP

- A20, BCL1-B20, 2-PK-3

M

Constitutive

Futagawa et al. , 2002

- BAL17

M

Negative

Futagawa et al. , 2002

Monocyte, Macrophage

- Primary

H

Constitutive

Laderach et al., 2003;

Ju et al., 2003; Jung

etal., 2004

-HL60

H

Constitutive

Ju etal., 2003

- RAW264.7, J774.1,

M

Constitutive

Pollok etal., 1994;

P388D1

Futagawa et al. , 2002

Dendritic cells

- Isolated from tonsil

H

Constitutive on subpopulation

Summers et al., 2001

- Derived from umbilical

H

Constitutive, enhanced by

Kim etal., 2002

cord blood progenitors

CD137 ligand

- Deriv. fr. monocytes or

H

Constitutive, enhanced by IL-1

Laderach et al., 2003

hematop. progenitors

and a-CD40

- Derived from monocytes

H

Const., enhanced by LPS,

Lee etal., 2003

TNF/ PGE2, IFN-y,

a-CD40, dsRNA

- Isolated from spleen

M

Const., enhanced by

Futagawa et al. , 2002

LPS/a-CD40

Sarcoma: Colon (Colo 205, H Constitutive Salih etal., 2000

HT29, HCT116), lung (LX 1, L2987), breast (SKBR), ovarian (A2780), prostate (PC3)

Sarcoma: Colon (Colo 205, H Constitutive Salih etal., 2000

HT29, HCT116), lung (LX 1, L2987), breast (SKBR), ovarian (A2780), prostate (PC3)

confirming that M-CSF is essential for monocyte survival and demonstrating that also CD137-induced monocyte survival is mediated by M-CSF.

Contrary to what might have been expected based on the life-prolonging effects of the CD137 ligand signal on monocytes, it also enhanced the rate of apop-tosis. Apoptosis as measured by the amount of fragmented DNA was several-fold increased throughout a 1 week culture period by CD137 ligand signaling. At the same time, the percentage of living cells was also increased (Langstein et al., 1999). Simultaneous induction of apoptosis, together with activation and proliferation is well known in lymphocytes as activation induced cell death (Green et al., 2003). However, this concept was not easily transferable to monocytes/macrophages as these cells have been assumed not to be able to proliferate. But when tested whether the CD137 ligand signal can induce proliferation it was indeed noticed that proliferation did occur. H3-thymidine incorporation increased steadily with time upon CD137 ligand signaling and at its peak at day 8 was increased by 30-fold. Close to 10% of the cells incorporated Bromo-deoxyuridine within a 1 h labeling interval, demonstrating that a large proportion of the cells were active in replicating their DNA (Langstein etal., 1999). But while DNA was replicated and the cells grew in size, their numbers did not increase implying that endomitosis occurred. Indeed, many of the cells contained several nuclei (Langstein et al., 1999).

Although induction of monocyte proliferation has also been described for M-CSF, the CD137 ligand signal is more potent in inducing proliferation and en-domitosis in monocytes, indicating that it induces the release of additional factors besides M-CSF. These are also induced in monocytes upon CD137 ligand signaling and are released as conditioned supernatants from CD137-treated monocytes transfer the proliferative potential to naive monocytes (Langstein et al., 1999).

This CD137 ligand-induced proliferation and endomitosis of monocytes overcompensates the loss of monocytes by CD137 ligand-induced apoptosis and allows the monocyte population to expand, not in numbers but in size.

Though CD137 can activate monocytes, it is less potent than classical monocyte activators such as LPS (Langstein et al., 2000). However, the CD137 ligand signal synergizes with LPS in activating monocytes since it is able to further increase cytokine release after maximum stimulation by LPS has been reached. Also, the combination of LPS and the CD137 ligand signal, but not either alone can induce myc expression in monocytes. (Langstein etal., 1998; Langstein etal.,

2000). It is in inducing proliferation that CD137 ligand exceeds the potency of other monocyte growth and activation factors and that may be its more important function in vivo.

The activating effects of CD137 ligand on monocytes have also been documented in vivo. Transgenic mice overexpressing CD137 ligand on antigen presenting cells (APC) develop a threefold increased number of macrophages (Zhu et al.,

2001). CD137 ligand-deficient mice on the other hand have an increased number of myeloid progenitor cells in the peripheral blood, bone marrow, and spleen (Kwon et al., 2002). It seems these cells cannot fully mature to monocytes, macrophages, and possibly dendritic cells due to the lack of the CD137 ligand signal.

CD137 ligand provides potent activating signals to monocytes and likely monocyte precursors. Since expression of CD137 is strictly activation-dependent, monocytes would encounter CD137 in tissues where activated immune cells or endothelial cells are present (Schwarz et al., 1995; Broll et al., 2001). Therefore, reverse signaling by CD137 ligand into monocytes is expected to amplify ongoing immune responses.

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