Association of AIM, a novel apoptosis inhibitory factor, with hepatitis via

supporting macrophage survival and enhancing phagocytotic function of

macrophages.

Ikuko Haruta1, Yoichiro Kato2, Etsuko Hashimoto1, Christina Minjares3, Shawna Kennedy3,

Hirofumi Uto3, Katsumi Yamauchi1, Makio Kobayashi2, Sei-ichi Yusa 4, 5, Urs Müller4, Naoaki

Hayashi1 and Toru Miyazaki3, 4.

1. Institute of Gastroenterology, and

2. Department of pathology, Tokyo Women’s Medical University. 8-1 Kawata-cho, Shinjuku-

ku, Tokyo. 162-8666 Japan.

3. Center for Immunology, The University of Texas Southwestern Medical Center.

6000 Harry Hines Blvd. NA7200. Dallas TX 75390-9093, USA.

4. The Basel Institute for Immunology. Grenzacherstrasse 487, CH-4005 Basel, Switzerland.

3. Fox-Chase Cancer Center. 7701 Burholme Avenue, Philadelphia, PA 19111, USA

Correspondence to: Toru Miyazaki.

Center for Immunology, The University of Texas Southwestern Medical Center.

(Tel) 1-214-648-7322 (Fax) 1-214-648-7331

(E-mail) toru.miyazaki@UTSouthwestern.edu

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Running Title

Function of AIM on macrophages at inflammatory regions.

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Summary

A hallmark of many inflammatory diseases is the destruction of tissue cells by

infiltrating hematopoietic cells including lymphocytes, neutrophils and

macrophages. The regulation of apoptosis of both target tissue cells and the

infiltrating cells is one of the key events that define the initiation and the

progression of inflammation. However, the precise picture of the apoptosis

regulation of the cells at the inflammatory sites is still unclear. We recently

isolated a novel apoptosis inhibitory factor, termed AIM, which is secreted

exclusively by tissue macrophages. In this report, we present unique

characteristics of AIM associated with liver inflammation (hepatitis), identified by

introducing an experimental hepatitis in both AIM-transgenic mice which

overexpress AIM level in the body, and normal mice. First, endogenous AIM

expression in macrophages is rapidly increased in response to inflammatory

stimuli. Second, AIM appears to inhibit the death of macrophages in the

inflammatory regions, judging by the remarkably increased number of

macrophages observed in the liver from transgenic mice. In addition, we show

that AIM also enhances the phagocytosis by macrophages, which emphasizes

the multifunctional character of AIM. All these findings strongly provoke an idea

that AIM may play an important role in hepatitis pathogenesis in a sequential

manner: first AIM expression is upregulated by inflammatory stimuli, and then in

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an autocrine fashion, AIM supports the survival of infiltrating macrophages as

well as enhances phagocytosis by macrophages, which may result in an efficient

clearance of dead cells and infectious or toxic reagents.

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Introduction

Apoptosis is a form of cell death, which is achieved by programmed signal

cascades triggered by differential stimuli depending on the variety of physiological

situations (1-4). Accumulating evidence has revealed that apoptosis plays a key role in

the pathology of inflammation (5-13). The present consensus is emerging that both

positive (inducing) and negative (inhibiting) regulation of apoptosis of tissue cells

influence the initiation and the progression of tissue damage (14-17). It has been

indicated that apoptosis at the inflammatory sites seems to be induced by: (1)

inflammatory cytokines produced by the infiltrating hematopoietic cells as well as the

tissue epithelium cells (18-20), and (2) interaction of CD95 (also called Fas) on the

tissue cells and CD95-ligand (CD95L) predominantly expressed on infiltrating T-

lymphocytes (21-27). In particular, the prevention of endotoxin-induced hepatitis by

the defect of CD95/CD95L ligation strongly implies that CD95-mediated apoptosis of

hepatocytes is the initial event in the process of hepatitis (21). In addition to apoptosis of

tissue cells, apoptosis regulation of infiltrating cells also seems to influence the

progression of inflammation. This indication was recently drawn by the observation of

non-obese diabetic (NOD) mice, a mouse model of human autoimmune diabetes (type I

diabetes), under CD95-deficient conditions, that were free from destruction of insulin-

producing pancreatic β cells by the infiltrating T-lymphocytes. In these mice, a

defect of CD95 appeared to decrease apoptotic death of T-lymphocytes

infiltrating into the pancreatic Langerhans islets, resulting in the prevention of

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tissue damage (28, 29). Thus, the regulation of both target tissue cells and

infiltrating cells influence the progression of inflammatory diseases.

However, we are essentially ignorant of the negative (inhibitory) regulation

of apoptosis at the inflammatory sites, though apoptosis must be regulated both

positively and negatively, and the balance of these two regulations may critically

influence the inflammation progression. This is mainly because the extracellular

ligands produced at the inflammatory sites that mediate inhibitory signals for

apoptosis, have not been well defined so far, despite the recent identification of

many intracellular apoptosis inhibitory elements (14-17).

We recently isolated a novel murine apoptosis inhibitory factor, termed AIM,

which is exclusively secreted by tissue macrophages including Kupffer cells in

the liver (30, 31). AIM inhibits apoptosis triggered by multiple stimuli including

irradiation, glucocorticoid and CD95-crosslinking (30). At the inflammatory sites

of most tissues, the macrophage is one of the cell types which are observed from

the very early stage of inflammation (32-36). Hence it is possible that AIM might

play a role in the negative regulation of apoptosis of cells at the inflammatory

sites.

In this report, we applied a mouse hepatitis model to address potential

involvement of AIM in the progression of inflammation in vivo. In addition, we

present a new AIM function associated with inflammation; enhancement of the

phagocytic function of macrophages, and discuss the multifunctional character of

AIM.

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Experimental Procedures

Mice.

Mice were bred and maintained in a specific pathogen free (SPF) animal facility at the

Basel Institute for Immunology (Basel, Switzerland) and a semi-SPF animal facility at

the Tokyo Women’s Medical University (Tokyo Japan). All animal experiments were

approved by the research Ethics Committee of the Tokyo Women’s Medical University.

Generation of transgenic mice.

AIM cDNA was subcloned into the pCAGGS expression vector (37). After the removal

of the vector sequence, a purified DNA fragment was microinjected into fertilized eggs of

(C57BL/6 x DBA/2)F1 mice. Embryos were transferred into the oviducts of CD-1

foster mothers. Founders were screened for transgene by PCR, and the resulting

transgenic founders were bred with C57BL/6 (B6) mice to generate progenies.

Transgene-positive progenies were backcrossed to B6 mice. After six backcrosses,

transgene positive and negative littermates were used for experiments.

Induction of endotoxin-induced fulminant hepatitis.

Mice were immunized by 1 mg/ mouse of heat inactivated Propionibacterium acnes (P.

acnes: Van Kampen Group, Inc., Utah USA) in 200 µL PBS by i.v. injection. 7 days

after the immunization, each mouse was challenged by i.v. injection of 2.5 µg

Lipopolysaccharide (LPS) in 150 µL PBS (38). At various time points after the

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LPS injection, mice were sacrificed, and blood and liver specimens were

collected to evaluate liver damage.

In situ mRNA analysis and histology.

The liver specimens were fixed in 10% formalin and embedded in paraffin. 4-µm

sections were cut out and placed on silane-coated slides. These sections were

subjected to in situ hybridization, using digoxigenin-labeled (Boehringer

Mannheim; Roche Molecular Biochemicals, Switzerland) antisense-AIM cDNA

probe (30, and refs therein). As a negative control, RNaseA (100µg/ml)

pretreatment was carried out prior to hybridization. After hybridization, sections

were treated with antidigoxigenine-alkaline phosphatase, and then signals were

developed by 4-nitroblue tetrazolium chloride (NTB) treatment.

The fixed liver tissues were also used for histological analysis of macrophage

detection. Sections were stained with BM-8 antibody (BMA Biochemicals,

Switzerland) which recognizes pan-monocyte/macrophages including the

Kupffer cells in the liver.

Phagocytosis assay.

1.5 x106 of either RAW264 cells (a mouse macrophage cell line (39), provided by

RIKEN Cell Bank, Tsukuba, Japan) or mouse peritoneal macrophage cells derived from

thioglycolate-stimulated C57BL/6 mice (30), were cultured for two hours with

appropriate numbers (cell:beads ratio was 25:1 according to the ref. 41) of FITC-labeled

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polystyrene latex beads (approximately 1.95µm in diameter; Sigma Chemical Co., St.

Louis, MO USA) in the presence or absence of recombinant AIM (rAIM). Culture

supernatant of the AIM-transfected Chinese ovarian carcinoma (CHO) cells was

used as a source of rAIM (30, 31). As a control, culture supernatant of non-

transfected CHO cells was used. After two hours incubation, cells were

collected by using cell scraper followed by an extensive wash with PBS, then

analyzed by fluorocytometry (FACScan; Becton & Dickinson).

Binding study.

Biotinylated rAIM protein and a control protein (a transcription factor localized in the

nucleus) generated by recombinant baculovirus-infected T. ni. Egg cells were used for

the binding study as we described in refs. 30 and 31. After the binding procedure of the

proteins to either peritoneal macrophages or RAW264 cells, cells were analyzed using a

FACScan cytometer (Becton Dickinson).

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Results

Induction of hepatitis strongly up-regulated AIM expression within

macrophage/Kupffer cells in the liver.

Although AIM is exclusively expressed by tissue macrophages, only part of the

macrophages in a tissue express AIM, suggesting the requirement of a specific

microenvironment surrounding the macrophages for AIM expression induction (30). The

precise mechanism for AIM expression regulation is, however, entirely unknown. In the

previous report, we demonstrated that AIM was strongly expressed within infiltrating

macrophages within BCG-induced granulomas, which may suggest potential up-

regulation of AIM expression in response to inflammatory stimuli (30). To test this

possibility, we introduced the endotoxin-induced hepatitis in C57BL/6 (B6) mice by

immunizing mice with P. acnes followed by LPS injection (38), and analyzed AIM

expression in both resident macrophages (Kupffer cells) and infiltrating macrophages.

Two hours after the LPS injection, although there was no obvious increase of BM-8

positive cells (compare Figure 1 a and c), most of these cells revealed AIM expression

when assessed by in situ mRNA analysis (Figure 1d). This was also confirmed

quantitatively by a northern blot analysis for AIM expression, by using RNA from the

liver of either before, or 2 hours after hepatitis induction (Figure 1g). Thus, hepatitis

induction rapidly up-regulated AIM expression in Kupffer cells. A markedly increased

number of BM-8 positive cells were observed in the liver after 12 hours, representing

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massive infiltration of macrophages into the liver (Figure 1e). Most of these cells also

expressed AIM strongly (Figure 1f). These results support the idea that inflammatory

stimuli rapidly induce AIM expression in macrophages.

Increased number of infiltrating macrophages in response to hepatitis induction in

AIM-transgenic mice.

Rapid up-regulation of AIM expression by hepatitis induction provokes a

potential involvement of AIM in hepatitis pathogenesis, either via inhibition of

apoptosis or other unknown function. In particular, since macrophages show a

remarkable binding capacity for AIM (30), and see Figure 5b this report), AIM

may function on macrophages at hepatitis regions in an autocrine fashion. To

obtain a clue for the possible role of AIM at inflammatory sites, we generated

transgenic mice overexpressing AIM under the control of chicken-beta actin

promoter and CMV enhancer (37) (Figure 2a). These mice overexpress AIM

ubiquitously, resulting in strikingly increased level of AIM concentration in the

serum (Figure 2b).

Despite the high AIM level in the serum, spontaneous infiltration of

macrophages was not obvious, judging by the comparable numbers of BM-8

positive cells in the liver of non-stimulated transgenic and negative littermate

mice (Figure 3a,b). This was also the case in all other tissues as assessed by a

histological analysis (data not shown). Thus AIM doesn’t appear to be involved

in either chemotaxis or migration-induction of cells in vivo. However, in

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response to hepatitis induction via endotoxin, transgenic mice apparently

harbored an increased number of infiltrating macrophages in the liver than did

control littermate mice (Figure 3c, d). In addition, many focal accumulations of

macrophages were observed in the liver of transgenic mice. This accumulation

was found at regions where the destruction of the liver tissue was apparent,

harboring marked infiltration of lymphocytes and neutrophils (Figure 3e).

Although these accumulations of inflammatory cells were also found in control

littermate mouse liver, the size and the number of them were remarkably larger in

transgenic mice.

At inflammatory sites, macrophages are exposed to many cytokines that

mediate apoptosis (18-20). Hence in AIM-transgenic mice, high dose of AIM

may support survival of macrophages at inflammatory sites, by inhibiting

apoptosis triggered by these cytokines, resulting in seemingly increased number

of infiltrating macrophages.

It is well known that CD95 (Fas)-mediated apoptosis of hepatocytes (liver

cells) is an essential event of hepatitis (21). It is not likely, however, that AIM

also protects hepatocytes against apoptosis, because the binding of AIM to

primary hepatocytes is not obvious so far tested by binding study using labeled-

recombinant AIM (data not shown). In line with this, there was no apparent

histological difference in the destruction of hepatocytes, in hepatitis induced-

transgenic and negative littermate mice (data not shown). Thus AIM appears to

be associated with hepatitis by supporting macrophage survival at inflammatory

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sites via apoptosis inhibitory effect, which may contribute to efficient clearance of

dead cells and infectious or toxic reagents by macrophages. However, in certain

tissues, AIM might be involved in regulating inflammation progression not only by

supporting macrophage survival, but also by preventing apoptosis of tissue cells.

The precise mechanism of AIM-mediated apoptosis inhibition is not clear yet.

As we described in the previous reports, there was no significant difference in

the expression levels of various apoptosis-inducing or inhibiting elements,

including c-FLIP, in thymocytes of AIM-/- and AIM+/+ mice, as well as in a

macrophage-cell line, J774.A1 in the presence or absence of recombinant AIM

(30, and our unpublished results), suggesting that AIM appears to inhibit

apoptosis of cells not simply by modulating the expression of these known

apoptosis-related molecules, but by mediating an independent signaling

cascade.

AIM enhances phagocytotic function of macrophages.

When analyzed histologically in a higher magnification, macrophages

harbored small nuclei within the cells, which probably represent nuclei from

phagocytised cells. Interestingly, significantly larger number of macrophages

harbored these small nuclei in AIM-transgenic mice than in negative littermate

mice. In addition, the number of these small nuclei per macrophage was

apparently larger in transgenic mice (Figure 4). Thus AIM may activate

phagocytotic function of macrophages.

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To test this hypothesis, we determined whether AIM enhances phagocytosis

by macrophages in vitro, by using recombinant AIM (rAIM) produced by AIM-

transfected CHO cells (30). Either peritoneal macrophages or a macrophage-

derived cell line, RAW264, both of which have binding capacity of AIM (see

Figure 5b), were incubated with FITC-labeled polystyrene latex beads in the

presence or absence of rAIM, and then the number of phagocytised beads in

each cell was analyzed by a fluorocytometory (40). Figure 5a shows the

histogram of FITC intensity from peritoneal macrophages that were incubated

either in the presence (plain line) or absence (dashed line) of rAIM. There are

several peaks, which represent the number of phagocytised beads in each cell.

The first peak (P0) corresponds to the macrophages that had no bead. This

population was far smaller in size when macrophages were incubated in the

presence of rAIM (2.9% in rAIM (+) v.s. 13.3% in rAIM (-)), indicating most of

macrophages incubated with rAIM phogocytised beads in two hours. In the

absence of rAIM, 27.1% of macrophages phagocytised a single bead (P1),

26.1% did two beads (P2), and 14.9% did three beads (P3) respectively. There

seemed to be a few macrophages, which had more than four beads (P4), though

they did not generate obvious peaks. In contrast, the major peak was P4 when

cells were incubated in the presence of rAIM: 69% of cells had more than four

beads (P4 in Figure 5a). Similar results were obtained when assessed by using

a macrophage-derived cell line, RAW264. Almost twice the number of cells

achieved phagocytosis of beads in two hours when incubated with rAIM (60% of

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incubated cells in rAIM (+) v.s. 34% of incubated cells in rAIM (-)). Furthermore,

the average of the intensity of FL-1 positive population was significantly higher in

rAIM+ population (rAIM(+) : rAIM(-) = 42 : 29), indicating each cell pahgocytised

larger number of beads in the presence of rAIM.

All together, AIM apparently enhances the phagocytotic function of

macrophages. However, rAIM revealed more efficient enhancement in

peritoneal macrophages than in RAW264 cells. This is, perhaps, due to the

difference of the binding capacity for AIM between the two cell types as shown in

Figure 5b, which clearly shows the significantly higher binding capacity of

peritoneal macrophages.

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Discussion

Inflammatory stimuli upregulate AIM expression in macrophages.

As we previously reported (30), AIM expression regulation harbors several

unique characteristics; (1) AIM expression is restricted in tissue macrophages,

(2) in a tissue, AIM expression is observed in only a part of the macrophages, (3)

when ex vivo macrophages are cultured on a plastic dish, AIM expression

entirely disappears, (4) AIM expression in macrophages is unable to be induced

in vitro by any reagents which are known to activate macrophages, including

PMA, LPS and various cytokines. These previous observations implied that a

specific microenvironment is required for AIM expression induction in

macrophages (30).

In the present study, we found that inflammatory stimuli appear to induce AIM

expression in vivo. Soon after hepatitis induction, most of resident macrophages

(Kupffer cells) and infiltrating macrophages in the liver revealed strong

expression of AIM. Since AIM expression had already been up-regulated in

Kupffer cells before infiltration of leukocytes was apparent, inflammatory

cytokines produced by the liver tissue cells as well as by Kupffer cells themselves

might predominantly induce AIM expression at the early stage of inflammation.

At the later stage of inflammation, cytokines produced by infiltrating lymphocytes

may also contribute to the upregulation of AIM expression. Nevertheless, since

AIM expression in ex vivo macrophages is not induced on a plastic dish in vitro

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even by various inflammatory cytokines (30), inflammatory stimuli may synergies

the basic mechanism of AIM expression induction, which may depend on

microenvironment, e.g. cell-cell interaction between specific type of cells and

macrophages. Further study will precisely clarify the elements required for AIM

expression induction.

AIM enhances phagocytosis of macrophages: multifunctional character of AIM.

We recently reported that AIM induces strong and sustained growth inhibition

of B-lymphocytes in combination with TGF-β1 (31). Thus, other than apoptosis

inhibitory effect, AIM has different functions depending on the target cell type,

and the combination with other cytokine(s). In line with this, we identified that

AIM enhances phagocytotic function of macrophages. Contrary to the case of

B-lymphocytes (31), AIM revealed two different functions on macrophages,

apoptosis inhibition and enhancement of phagocytosis, without specific

conditions. This multi-functional character of AIM is reminiscent of various

cytokines such as IL-4, which activates B-lymphocyte proliferation in

combination of IgM cross-linking, as well as induces immunoglobulin class

switching towards IgE (41).

So far, a variety of reagents are known to activate phagocytotic function of

macrophages, including cytokines also secreted from macrophages, such as G-

CSF, GM-CSF, IL-4 and IL-10 (42, 43). One might argue that enhancement of

phagocytosis may be an indirect effect; AIM might induce production of such

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cytokines in macrophages, resulting in enhanced phagocytosis. It is, however,

unlikely because we could not detect any up-regulation of expression of these

cytokines in macrophages by AIM in vitro (manuscript preparation), though we

cannot mutually exclude a possibility that AIM may induce unknown factor(s)

which may enhance phagocytosis.

Perspective

It may be worth emphasizing that the unique characteristics of AIM which we

presented in this report strongly imply the association of AIM with hepatitis in a

sequential manner: first AIM expression is up-regulated in response to

inflammatory stimuli, and then, in an autocrine fashion, AIM supports the survival

of infiltrating macrophages, as well as activates phagocytosis by macrophages,

which may result in an efficient clearance of dead cells and infectious or toxic

reagents.

Acknowledgements

Authors thank to Dr. Sumiko Yamakawa and Itoe Okamoto Foundation for

financial support, Dr. Shin Ohnishi (Tokyo, Japan) for discussion. The Basel

Institute for Immunology was founded and supported by Hoffmann-La Roche

Ltd, Basel Switzerland.

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Figure Legends.

Figure 1: Rapid up-regulation of AIM expression by hepatitis induction.

In situ hybridization analysis of AIM expression (violet/ dark blue signals in b, d, and f)

and its comparison with immunolocarization of pan-macrophage (including Kupffer

cells) antigen, BM-8 (dark yellow/ brown signals in a, c, and e) in the liver from mouse

before (a, b), 2 hours after (c, d) and 12 hours after (e, f) inducing fulminant hepatitis.

(a, b) AIM is expressed in few Kupffer cells. (c, d) Two hours after hepatitis induction,

far increased number of Kupffer cells expressed AIM. (e, f) Twelve hours after hepatitis

induction, large number of macrophages infiltrated in the liver (e), and they expressed

AIM (f). Magnifications: a, c, e, x100; b, d, f, X150.

(g) Northern blot analysis for AIM expression. Total Liver RNA was obtained from

either pre- or 2 hours after-hepatitis induction. 10µg of RNA was separated on an

agarose gel, blotted, and hybridized with a 32P-labeled either AIM or mouse β-

actin cDNA probe.

Figure 2: AIM transgenic mice.

(a) Transgene construct. A 1.0Kb fragment of the coding sequence (cds) of AIM cDNA

was subcloned into the pCAGGS expression vector, which contains CMV-enhancer,

chicken β-actin promoter and rabbit β globin exon and intron (37). (b) Western

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blot analysis of serum from AIM-transgenic mouse or negative littermate mouse

for AIM. 5µl of serum from each type of mouse, as well as 500ng of

recombinant AIM protein produced by recombinant baculovirus-infected T. ni.

Egg cells were size-fractionated on 10% SDS-PAGE, blotted on a membrane,

and stained by anti-AIM monoclonal antibody (clone 3G14, ref. 20). Signals

were developed by the ECL system (Amersham Pharmacia Biotech).

Figure 3: Potentially longer survival of inflammatory macrophages in AIM-

transgenic mouse liver.

Liver sections from AIM-transgenic mouse (a, c) and negative littermate mouse

(b, d) either before or 8 hours after hepatitis induction were stained by BM-8

(dark yellow/ brown signals) for pan-macrophage (including Kupffer cells)

antigens. There is no increase of BM-8 positive cells in transgenic mouse liver

before hepatitis induction (a, b). 8 hours after hepatitis induction, markedly

larger number of BM-8 positive cells were observed in transgenic mouse liver

than in negative littermate mouse liver (c, d). In transgenic mouse liver, a

number of accumulation of BM-8 positive cells and leukocytes were detected (e).

Magnifications: a-d, x100; e, x200.

Figure 4: enhanced phagocytosis by macrophages in AIM-transgenic mouse

liver.

Liver sections from AIM transgenic mouse (a) and negative littermate mouse (b,

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c) of 8 hours after hepatitis induction were stained for BM-8, and analyzed in a

high magnification (x400). A number of BM-8 positive cells which contained

more than 2 small nuclei derived from dead cells were detected in transgenic

mouse liver (a). In contrast, BM-8 positive cells in negative littermate mouse

contained 0 or 1 small nucleus derived from dead cells (b, c).

Figure 5: AIM accelerates phagocytotic function of macrophages in vitro.

(a) Peritoneal macrophages were incubated with FITC-labeled polystyrene latex

beads for 2 hours in the presence or absence of recombinant AIM (rAIM). Cells

were then analyzed for their uptake of the beads by FACS can (Becton-

Dickinson). P0-P3 correspond to the number of phagocytized beads (e.g. P3=3

beads uptake) respectively, and cells which have more than 4 beads together are

represented as P4. In the presence of rAIM (Plain line), macrophages

apparently showed enhanced phagocytosis of beads as compared to cells in the

absence of rAIM (Dashed line).

(b) AIM binding capacity of peritoneal macrophages and a macrophage cell line,

RAW264, are presented as log fluorescence by black histograms. And the control

protein bindings that are regarded as backgrounds are represented by white

histograms. Peritoneal macrophages showed higher capacity of AIM binding

than RAW264 cells.

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Hayashi and Toru MiyazakiHirofumi Uto, Katsumi Yamauchi, Makio Kobayashi, Sei-ichi Yusa, Urs Müller, Naoki Ikuko Haruta, Yoichiro Kato, Etsuko Hashimoto, Christina Minjares, Shawna Kennedy,

macrophage survival and enhancing phagocytotic function of macrophagesAssociation of AIM, a novel apoptosis inhibitory factor, with hepatitis via supporting

published online April 9, 2001J. Biol. Chem. 

  10.1074/jbc.M100324200Access the most updated version of this article at doi:

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