ORIG INAL ART ICLE

Distinct granuloma responses in C57BL ⁄6J and BALB ⁄cByJ mice inresponse to pristaneHuaiyong Chen*, Dongmei Liao*, Derek Cain*, Ian McLeod*, Yoshihiro Ueda*, Ziqiang Guan�,Christian Raetz� and Garnett Kelsoe**Department of Immunology, Duke University Medical Center, Durham, NC, USA and

�Department of Biochemistry, Duke

University, Durham, NC, USA

Granuloma formation constitutes a protective cellular reac-

tion in response to persistent microbial antigens including

Mycobacterium tuberculosis (Meyer et al. 1975), eggs of the

blood fluke Schistosoma mansoni (Warren & Domingo

1970), and numerous pathogenic fungi (Hauser & Rothman

1950; Ley et al. 1951). The granuloma response sequesters

foci of microbe pathogens, preventing their dissemination

and restricting inflammation to protect surrounding tissue

(Co et al. 2004). Granulomas can also be induced by foreign

bodies resistant to catabolism, such as implanted biomateri-

als (Zeller 1983), sand particles (Ginsberg & Becker 1951),

and coal dust (Kido et al. 1995).

The laboratories of Potter and Reeves have independently

demonstrated that oil granulomas are readily induced in

BALB ⁄ c mice by intraperitoneal injections of pristane, a nat-

urally-occurring, saturated alkane (2,6,10,14-tetramethylpen-

tadecane) (Potter & Maccardle 1964; Nacionales et al.

2006). The common history of pristane-induced granuloma

responses in BALB ⁄ c mice has been well documented: a few

days after injection, small volumes of pristane are phagocy-

tosed by macrophages (M/) while larger volumes of pristane

become surrounded by inflammatory leucocytes to form oil-

cell aggregates that adhere to peritoneal surfaces, especially

the mesentery (Potter & Maccardle 1964). Eventually, the

mesothelium grows over the oil-cell aggregates to form oil

granulomas that accumulate on mesenteric surfaces as long

as free oil is available (Potter & Maccardle 1964).

In addition to M/, BALB ⁄ c pristane granulomas contain

lymphocytes, neutrophils, and plasma cells (Potter & Mac-

cardle 1964) that are recruited both from the PC and from

the mesenteric blood supply (M. Potter, unpublished data).

Cellular responses to peritoneal pristane vary significantly

between inbred mouse strains. For example, BALB ⁄ cJ but

not C57BL ⁄ 6J, develop arthritis (Wooley et al. 1989) and

50–60% of BALB ⁄ cAn mice develop peritoneal plasmacyto-

mas (PCT) within a year of pristane injection (Potter 2003).

In contrast, C57BL ⁄ 6J do not develop arthritis (Wooley

et al. 1989) and only 5% of C57BL ⁄ 6J mice eventually

INTERNATIONAL

JOURNAL OF

EXPERIMENTAL

PATHOLOGY

doi: 10.1111/j.1365-2613.2010.00725.x

Received for publication:26 February 2010Accepted for publication: 2 June 2010

Correspondence:Garnett KelsoeDepartment of ImmunologyDuke UniversityDurhamNC 27710USATel.: +1 919 613 7815Fax: +1 919 613 7878E-mail: ghkelsoe@duke.edu

Summary

Granuloma formation is an inflammatory response of the host against invading

pathogens or indigestible substances. We generated mesenteric oil granulomas by

injecting pristane into the peritoneal cavity (PC) of mice, and compared oil granu-

loma formation in the C57BL ⁄ 6J and BALB ⁄ cByJ strains of mice. The formation

and kinetics of oil granulomas were distinct between the two strains. In C57BL ⁄ 6J

mice, injected pristane induced oil granuloma formation at both the mesenteric cen-

ters (MG) and margins (SG). MG was resolving by 11 weeks, and SG persisted. In

BALB ⁄ cByJ mice, MG developed slower but persisted longer than in C57BL ⁄ 6J mice,

and SG resolved sooner than in C57BL ⁄ 6J mice. Injection of India ink revealed that

phagocytes were localised mainly to the SG in C57BL ⁄ 6J mice, but were located dif-

fusely in both MG and SG of BALB ⁄ cByJ mice. SG cells expressed more monocyte

chemotactic protein-1 (MCP-1) mRNA than MG cells in C57BL ⁄ 6J mice, but there

was no difference in MCP-1 expression between the MG and SG in BALB ⁄ cByJ

mice. These observations suggest that the recruitment of inflammatory leucocytes

under the direction of chemokines differentiates the patterns of granuloma responses

to pristane in C57BL ⁄ 6J and BALB ⁄ cByJ mice.

Keywords

MCP-1, oil granuloma, pristane, strain

Int. J. Exp. Path. (2010), 91, 460–471

460 � 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd

develop PCT (Potter 2003). The genetic differences responsi-

ble for these disparate responses are poorly understood, in

part because the induction and resolution of pristane granu-

lomas in resistant strains has not been detailed.

Active granulomas are essential for BALB ⁄ cAn PCT

induction and persistence, as most primary PCT do not sur-

vive when transplanted into normal, syngeneic hosts but do

grow in pristane-conditioned recipients (Potter et al. 1972).

Nordan et al. (1989) first noted exceptional amounts of

interleukin 6 (IL-6) in BALB ⁄ cAn granuloma M/ and subse-

quent studies in IL-6 knockout animals demonstrated resis-

tance to PCT induction by pristane (Lattanzio et al. 1997).

IL-6 is, therefore, a crucial factor in PCT transformation. In

addition to IL-6, other factors control PCT induction by

pristane, including constitutive expression of the anti-apop-

totic factors Bcl2 and Bcl-xL (Potter 2003; Silva et al. 2003).

Reciprocally, two unidentified loci on chromosome 4 have

been shown to mediate the resistance of DBA ⁄ 2 mice to

PCT induction (Potter et al. 1994).

Mesenteric granulomas (MG) are considered to be the

cellular and environmental source of pristane-induced PCT

(Potter & Maccardle 1964). Whereas the formation of pris-

tane granulomas has been detailed in BALB ⁄ c mice (Potter

& Maccardle 1964), granuloma induction in PCT resistant

strains, including C57BL ⁄ 6J, is poorly understood. To bet-

ter understand why C57BL ⁄ 6J mice are resistant to PCT,

we studied the evolution of pristane granulomas in

C57BL ⁄ 6J mice in comparison to the sensitive BALB ⁄ cByJ

strain. We found that pristane induces vigorous granuloma

responses in both mouse strains but that the types of granu-

lomatous tissue formed in these mice are distinct. In

C57BL ⁄ 6J mice, pristane results in the accumulation of

prominent serosal granulomas (SG) at the interface of the

mesenteric margins and gut; in contrast, BALB ⁄ cByJ

animals respond with a centripetal distribution of MG.

Distinctive expression patterns of monocyte chemotactic

protein-1 (MCP-1), a M/ attracting chemokine (Mantovani

et al. 1993), is suggested to account for the major differ-

ences in granuloma development in C57BL ⁄ 6J and BALB ⁄cByJ mice, and we propose that the distribution of pristane

granulomas on the mesentery and ⁄ or within the PC directs

the distinct pathologies induced by pristane in C57BL ⁄ 6J

and BALB ⁄ cByJ mice.

Materials and methods

Mice and treatments

Male and female C57BL ⁄ 6J and female BALB ⁄ cByJ mice

were purchased from Jackson Laboratories (Bar Harbor,

ME, USA) and housed at the Duke University Animal Care

Facility with sterile bedding, water, and food. To minimise

any effects of incidental antigen exposure on granuloma for-

mation, all mice used in this study were maintained under

specific pathogen free conditions. At 7–9 weeks of age, mice

received a single intraperitoneal injection of pristane (100 or

300 ll; 2.8 · 10)4 or 8.3 · 10)4 mol respectively) (‡95%

pure, Sigma-Aldrich, St Louis, MO, USA). Age-matched,

untreated mice were used as controls.

In a separate study of India ink uptake by the mesentery,

0.5 ml of a 1 ⁄ 10 dilution of India ink in PBS was injected

i.p. into mice that had been given 300 ll pristane 3 weeks

earlier. Mice were sacrificed and various tissues recovered

for analysis at 4 h, 1 day, 4 days, and 12 days after injec-

tion.

All experiments involving animals were reviewed and

approved by the Duke University Institutional Animal Care

and Use Committee.

Tissue isolation and cell collection

Whole mesenteric tissue (WMT), from the distal duodenum

through terminal ileum, was removed intact by dissection

from naı̈ve or pristane-treated mice. WMT was spread out

in ice-cold PBS and photographed with a Canon camera

(EOS 20D) equipped with a macro-lens (EF-S 60 mm). Sin-

gle cell suspensions were prepared by digesting recovered tis-

sue in RPMI 1640 medium containing 0.5 mg ⁄ ml type I

collagenase (Sigma-Aldrich), 0.5 mg ⁄ ml type IV collagenase

(Sigma-Aldrich), 0.2 mg ⁄ ml deoxyribonuclease I (DNase I,

Sigma-Aldrich), and 25 mM HEPES buffer in Erlenmeyer

flasks with constant stirring. The tissue digestion was carried

out at room temperature for 1 h; the resulting cell suspen-

sion was removed and filtered through fine nylon mesh

(Denville Scientific Inc., Metuchen, NJ, USA). Cells were

then washed and resuspended in buffer (HBSS+ 5% FBS) for

flow cytometric analysis or chemokine expression analysis

by quantitative PCR. In some experiments, 5 mm biopsy

punches from the MG of pristane-treated mice or the corre-

sponding area in naı̈ve mice were excised. Individual SG in

pristane-treated mice and the corresponding area in naı̈ve

mice were removed separately by a 2 mm punch. Cells in

these tissues were extracted using collagenase ⁄ DNase I as

for WMT.

Resident PC cells were collected by lavage with 10 ml of

ice-cold RPMI supplemented with 5% FBS. After centrifuga-

tion, cell pellets were harvested for flow cytometric analysis.

Cell samples were also obtained from the spleen and right

femur for flow cytometric comparison to PC cell and popu-

lation analyses.

Flow cytometry

Cells harvested from lymphoid tissues or the PC were incu-

bated with ammonium chloride buffer for 1 min on ice to

lyse red blood cells (RBCs) before immunolabelling. Typi-

cally, approximately 106 nucleated cells were incubated with

FcR (CD16 ⁄ 32) blocking antibody for 20 min, washed, and

then labelled (15 min ⁄ ice) with antibodies specific for B220

(PE-Cy7), TCRb (APC), CD11c (PE), Gr-1 (FITC), and

CD11b (APC-Cy7). For B-cell subset analysis, cells were

stained with antibodies specific for B220 (PE-Cy7), CD93

(APC), and IgD (PE). B1 cells were identified by labelling

cells with B220 (PE-Cy7), CD5 (PE), IgM (TxR), and

Strain-specific granuloma responses 461

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

CD11b (APC-Cy7). CD138 (PE) antibody was used to iden-

tify plasma cells in tissues. Propidium iodide (Sigma-Aldrich)

was included to identify dead cells. Labelled cells were anal-

ysed in a FACSVantage with DIVA option. Flow cytometric

data were analysed with FlowJo software (Tree Star,

Ashland, OR, USA).

Quantitative PCR

Total RNA was extracted from approximately 106 cells in

TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Messenger

RNA was reverse transcribed (Superscript III; Invitrogen)

with oligo (dT) primer for 1 h at 50 �C. Quantitative PCR

was performed in an iCycler thermal cycler (Bio-Rad Labo-

ratories, Hercules, CA, USA) with SYBR� Green PCR core

reagents (Applied Biosystems, Foster City, CA, USA) and

primers for specific genes. Amplification conditions were as

follows: denaturation at 94 �C ⁄ 10 min; amplification at

94 �C ⁄ 15 s, 60 �C ⁄ 45 s, 40 cycles. These primers were

used: b-actin, forward, 5¢-AGCCATGTACGTAGCCATCC-

3¢, and reverse, 5¢-CTCTCAGC TGTGGTGGTGAA-3¢;MCP-1, forward, 5¢-CTTCTGGG CCTGCTGTTCA-3¢, and

reverse, 5¢-CCAGCCTACTCATTG GGATCA-3¢; TNFa,

forward, 5¢-TCCCCAAAG GGATGAGAAGTTC-3¢, and

reverse, 5¢-GGGAGTAGACAAGGTACAAC-3¢; IL-6, for-

ward, 5¢-CCCAACAGACCTGTCTAT ACC-3¢, and reverse,

5¢-CAGCTTATCTGTTAGGAGAGC-3¢. The relative levels

of mRNA for specific target genes were calculated by the

comparative Ct (threshold cycle) method recommended by

the manufacturer (Applied Biosystems) normalised to b-

actin message in the same sample. In brief, specific DCt

values were determined as [DCt = (Ctb-actin)–(Cttarget)]; rela-

tive expression levels were defined as: 2)DCt.

Immunohistochemistry and immunofluorescence staining

Granulomatous tissues were excised en bloc, rinsed in

media, and snap frozen in OCT embedding compound.

Cryostat sections were cut at 5 lm, fixed in acetone ⁄ metha-

nol (1:1 vol.) for 10 min at )20 �C, and air dried. Slides

were then washed in PBS buffer supplemented with 0.5%

BSA and 0.1% Tween-20, then blocked with purified rat

IgG and ⁄ or antibody to mouse CD16 ⁄ 32 (1:100, BD

Pharmingen, San Jose, CA, USA) for 1 h at room tempera-

ture. For immunofluorescence staining, sections were then

stained in humidified boxes with PE-conjugated CD138

antibody (1:400, BD Pharmingen) for 3.5 h at room temper-

ature. DAPI (1 lg/ml, Fluka, Buche, Switzerland) was

included to identify nuclei. Sections were washed and

mounted in Fluoromount-G. In a separate experiment, sec-

tions were incubated with biotin-conjugated CD11b anti-

body, followed by streptavidin-conjugated horseradish

peroxidase (HRP). For haematoxylin and eosin (H&E)

staining, SG and MG were harvested as described for immu-

nofluorescent staining, and fixed in Fekete’s modification of

Tellyesniczky’s fixative [70% ethanol:formalin:glacial acetic

acid (20:2:1 vol.)] overnight at 4 �C. Fixed tissue was

embedded in paraffin, cut in 5 lm sections, and processed

for H&E staining.

Statistics

Differences between paired groups were analysed using Stu-

dent’s t-test; P values £ 0.05 were considered significant.

Results

Distinct mesenteric responses to pristane in C57BL ⁄ 6Jand BALB ⁄ cByJ mice

To induce a granuloma response, 100 or 300 ll pristane

was administered i.p. to C57BL ⁄ 6J or BALB ⁄ cByJ mice,

which were then sacrificed at various intervals (day 3–week

11). The gut-associated WMT, from distal duodenum to ter-

minal ileum, was excised and photographed. Pristane

induced the formation of two classes of granulomas on the

mesentery: granulomas about the center of the mesenteric

tissue and away from peripheral fat and blood vessels (MG);

and individual SG that developed along the border of the

mesentery and intestine. Granuloma responses to pristane

were dose dependent; in mice treated with 100 ll pristane,

granulomas resolved within 8–11 weeks, whereas injection

of 300 ll pristane resulted in granulomas that persisted

beyond 11 weeks (Figure S1). In all subsequent experiments,

mice were injected with 300 ll pristane.

Following pristane injection, the clear mesentery in

C57BL ⁄ 6J mice turned milky, thickened as early as day 3,

and became more dense by week 3 (Figures 1a and S1). MG

began resolving at week 11, when the mesenteric tissue

appeared much clearer and patchy. SG development began

at week 2 after pristane (Figure S1), and was well-formed by

week 3, with an average of 41(± 12) [�x (± SD)] SG per

mouse (Figure 1b). At week 3, SG were typically 1–2 mm in

diameter but continued to grow in number until the margins

of individual SG became indistinguishable from those of

neighbouring SG (week 11). The number of SG at week 11

was 57(± 7) per mouse.

Granulomas responses of BALB ⁄ cByJ mice to pristane fol-

lowed closely the earlier description on BALB ⁄ cAn strain

(Potter & Maccardle 1964) and the MG of BALB ⁄ cByJ

mice exhibited distinctive structure and distributions in

comparison to those in C57BL ⁄ 6J mice, evidenced by focal

leukocytic infiltrations. Similar to Dr. Potter’s observations

in BALB ⁄ cAn mice (Potter & Maccardle 1964), polyps were

found on the mesentery of BALB ⁄ cByJ mice in response to

pristane (Figure 1a). By week 11, the mesenteric deposition

of pristane progressed dramatically, with numerous plaques

of various size on the mesentery (Figure 1a). Polyps were

not found at this time point. SG were rare in BALB ⁄ cByJ

mice; only 6(± 3) SG per mouse were observed at week 3

and no SG were present at week 11 (Figure 1b). We con-

clude that the resolution of the granuloma response to pris-

tane is distinct in these resistant and sensitive mouse

strains.

462 H. Chen et al.

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

Nonetheless by histology, the general structures of MG

and SG were similar in both C57BL ⁄ 6J and BALB ⁄ cByJ

mice. Both MG and SG contained randomly distributed leu-

cocytes. Typically, pristane was trapped in both MG and

SG, appearing as oil droplets of various sizes (Figure 1c). It

was our impression that the cellular density of granulomas

in BALB ⁄ cByJ mice was somewhat lower than in C57BL ⁄ 6J

animals and that BALB ⁄ cByJ MG and SG contained more

but smaller oil droplets (Figure 1c).

Comparable SG cellularity but distinct MG populationsin C57BL ⁄ 6J and BALB ⁄ cByJ mice

To quantify the cellularity of pristane-induced granulomas

in C57BL ⁄ 6J and BALB ⁄ cByJ mice, cells were isolated by

collagenase digestion of WMT, which included both SG and

MG, from naı̈ve mice or pristane-treated mice. Cells were

counted, and the types of leucocytes found in the WMT

were identified by antibodies specific for B220, TCRb,

CD11c, CD11b, and Gr-1. Naı̈ve C57BL ⁄ 6J WMT con-

tained few conventional B cells (B220+), T cells (TCRb+),

dendritic cells (DCs, CD11c+), neutrophils (CD11b+Gr-1hi)

and M/ (CD11b+Gr-1low) (Figure 2a).

In normal mesenteric tissue, the combination of these leu-

cocyte populations comprised <10% of the mesenteric tissue.

After pristane, however, the mesentery was reshaped by a

considerable influx of inflammatory leucocytes. The total

number of cells in naı̈ve C57BL ⁄ 6J mesentery was

3.9(± 2.3) · 105 cells ⁄ mouse but 3 weeks after pristane

injection, the total cell number increased 60-fold

[2.4(± 0.2) · 107 cells ⁄ mouse, Figure 2b] and remained ele-

vated through week 11 [2.2(± 0.6) · 107 cells, Figure 2b].

Whereas naı̈ve BALB ⁄ cByJ and C57BL ⁄ 6J WMT exhibited

comparable leukocytic cellularities [3.7(± 1.9) · 105 cells],

the influx of leucocytes in response to pristane was dis-

tinct. At week 3 after pristane, BALB ⁄ cByJ cellularity

[6.9(± 3.7) · 106 cells] was only 25% of that in C57BL ⁄ 6J

mice (P < 0.001) but by week 11 BALB ⁄ cByJ WMT cellular-

ity surpassed [3.9(± 0.8) · 107 cells], which was 75%

greater than that in C57BL ⁄ 6J WMT at the same time point

(P < 0.05). The absolute numbers of M/, T cells, and DCs

in BALB ⁄ cByJ WMT were <30% of those in C57BL ⁄ 6J

WMT at week 3 (P < 0.01), although neutrophil numbers

were modestly but not significantly higher in BALB ⁄ cByJ

WMT compared to C57BL ⁄ 6J WMT (Figure 2b). Similarly,

there was no difference in B-cell numbers between C57BL ⁄6J and BALB ⁄ cByJ WMT at week 3 (Figure 2b). In C57BL ⁄6J mice, the numbers of each leucocyte population in WMT

stayed the same or decreased from weeks 3 to 11 after pris-

tane injection (Figure 2b). In contrast, the numbers of each

cell type in BALB ⁄ cByJ WMT continued to increase so that

they were similar to, or even slightly greater than, that in

C57BL ⁄ 6J WMT by week 11 (Figure 2b). These data show

that C57BL ⁄ 6J and BALB ⁄ cByJ differ in their recruitment

of leucocytes to the WMT during the pristane-induced

granuloma response.

To determine if these differences in leucocyte recruitment

were due to a decreased frequency of cells in the MG of

(a)

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C57BL/6JBALB/cByJ

MG

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After pristane

Oil

SG

MG

Oil

Oil

OilOil

(b)

(c)

Figure 1 Differential response of themesentery to pristane in C57BL ⁄ 6J andBALB ⁄ cByJ mice. (a) After i.p. injectionof 300 ll (8.31 · 10)4 mol) of pristane,C57BL ⁄ 6J and BALB ⁄ cByJ mice weresacrificed at week 3 or week 11, andgut associated WMT from naı̈ve or pris-tane-primed mice was photographed. Awhite arrow indicates a polyp on mes-entery. (b) SG were numerated inC57BL ⁄ 6J and BALB ⁄ cByJ mice.***P < 0.001 shows the differencebetween C57BL ⁄ 6J mice vs. BALB ⁄ cByJmice. (c) An equivalent area of MG orstrip of SG was excised from eitherC57BL ⁄ 6J or BALB ⁄ cByJ mice at week3 or 11 after pristane, and the tissuewas fixed and stained with hematoxylinand eosin solution. Representative pic-tures are shown. Original magnification(objective): ·63.

Strain-specific granuloma responses 463

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

BALB ⁄ cByJ mice, the MG or its corresponding areas in

naı̈ve mice were excised and digested with collagenase to

extract cells from tissue. The frequency of cells in the

C57BL ⁄ 6J MG at week 3 was 1.1(± 0.4) · 104 cells ⁄ mm2 of

mesentery (Figure 2c), 80-fold greater than that of the corre-

sponding area of mesentery in naı̈ve C57BL ⁄ 6J mice

[1.3(± 0.1) · 102 cells ⁄ mm2]. The cellularity of BALB ⁄ cByJ

MG at week 3 after pristane was also elevated [2.0(± 0.5) ·103 cells ⁄ mm2] compared to the corresponding area in

naı̈ve BALB ⁄ cByJ mice [2.1(± 0.3) · 102 cells ⁄ mm2], but

was < 20% of that of C57BL ⁄ 6J MG at the same interval

(panel far left, Figure 2c). The frequency of each leucocyte

103

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Figure 2 Leucocyte infiltration. (a) Identification of cell types by flow cytometry. Single cell suspensions prepared from spleen of pris-tane injected C57BL ⁄ 6J mice (300 ll pristane, 11 weeks) were stained and analysed by flow cytometry as described in Materials andmethods. Live cells were positively selected for analysis of CD11c expression, which results in identification of CD11c+ DCs andCD11c- fraction. Analysing expression of B220 and TCRb of CD11c- cells reveals TCRb+ fraction (T cells) and B220+ fraction (con-ventional B cells). The CD11c-B220-TCRb- fraction was furthered fractionated by expression of CD11b and Gr-1 into CD11b+Gr-1low (monocytes) and CD11b+Gr-1hi (neutrophils). Monocytes, after migrating into tissues, are called M/. M/ in mesenteric tissuewere identified as CD11b+Gr-1low. (b) Cellularity of WMT was analysed by flow cytometry. (c) MG was punched out with a 5-mmpunch and its cellularity was determined as for the cellularity of WMT. Data represent the number of total cells of each cell type per1 mm2 of MG. (d) 4–8 Individual SG were isolated with a 2-mm punch at week 3 after injection of pristane, and digested with colla-genase. Cells were enumerated. The SG cellularity is presented as cell number ⁄ SG. Black diamonds or columns represent C57BL ⁄ 6Jmice, and white diamonds or columns represent BALB ⁄ cByJ mice. *P < 0.05; **P < 0.01; ***P < 0.001 shows the differencebetween C57BL ⁄ 6J mice vs. BALB ⁄ cByJ mice.

464 H. Chen et al.

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

population was significantly less in BALB ⁄ cByJ MG than in

C57BL ⁄ 6J MG (Figure 2c). From weeks 3 to 11 after pris-

tane injection, the dynamic of MG cellularity in C57BL ⁄ 6J

mice was reciprocal to that in BALB ⁄ cByJ mice. Over this

period, the numbers of each cell type decreased in

C57BL ⁄ 6J mice, but increased in BALB ⁄ cByJ mice (Fig-

ure 2c) such that, by week 11 after pristane, the frequency

of each cell type was significantly higher in BALB ⁄ cByJ MG

than in C57BL ⁄ 6J MG (Figure 2c).

We also analysed pristane-induced SG in C57BL ⁄ 6J and

BALB ⁄ cByJ mice. Three weeks after pristane, several indi-

vidual SG or their corresponding areas in naı̈ve mice were

isolated from C57BL ⁄ 6J and BALB ⁄ cByJ mice. The cellu-

larity of SG was comparable between C57BL ⁄ 6J [1.2(± 0.3)

· 105 cells ⁄ SG] and BALB ⁄ cByJ mice [1.4(± 0.6) · 105

cells ⁄ SG] (Figure 2d) as were the numbers of each leuco-

cyte type (Figure 2d). The cellularity of SG at week 11

after pristane could not be compared as no SG were

observed in BALB ⁄ cByJ mice at this time point (Figure 1b).

Collectively, these data demonstrate that the WMT cellu-

larity attributable not only to the number of SGs but also

to the frequency of cells in MG.

Pristane increases non-adherent cell numbers in theperitoneum

The development of pristane-induced granulomas requires

the continuing adhesion of alkane micelles surrounded by

organised collections of peritoneal cells (Potter & Maccardle

1964). The numbers of peritoneal cells available to enter

these complexes may, therefore, affect granuloma develop-

ment. To determine if the differences in WMT cellularity

between C57BL ⁄ 6J and BALB ⁄ cByJ mice could be attribut-

able to differences in the availability of free peritoneal cells,

non-adherent peritoneal cells were washed out after pristane

injection and subsequently enumerated and characterised by

flow cytometry. The number of free peritoneal cells increa-

sed dramatically 3 weeks after pristane injection into both

C57BL ⁄ 6J and BALB ⁄ cByJ mice compared to naı̈ve mice.

By 3 weeks after injection into C57BL ⁄ 6J mice, pristane

induced striking influxes of neutrophils and M/ (together

constituting >80% of leucocytes) into the PC, and modest

increases in the numbers of T cells and DCs (Figure 3a).

BALB ⁄ cByJ exhibited similar increases in the numbers of PC

neutrophils, M/, T cells and DCs (Figure 3a). The number

of Conventional B cells in the peritoneal exudates of naı̈ve

C57BL ⁄ 6J mice was approximately six times that in

BALB ⁄ cByJ mice. Three weeks after pristane, B cell numbers

were decreased in C57BL ⁄ 6J mice, but were significantly ele-

vated in BALB ⁄ cByJ mice (Figure 3a). By week 11 after pris-

tane, the numbers of neutrophils, M/, DCs, T cells, and

conventional B cells decreased to very low levels in

C57BL ⁄ 6J mice, but remained elevated in BALB ⁄ cByJ mice

(Figure 3a). These results imply that generalised peritoneal

inflammation continues in BALB ⁄ cByJ mice 11 weeks after

pristane, but is resolved, along with the focal granuloma

response (Figure 1a), in C57BL ⁄ 6J mice.

Characterisation of granuloma B cells

B1 cells, comprising B1a (B220lowCD5+IgMhiCD11blow) and

B1b (B220lowCD5-IgMhiCD11blow) cells (Figure 3b), are a

constitutive population of the PC and, presumably, the

source of pristane-induced PCT (Potter 2003). Three weeks

after pristane, there were significant reductions in the num-

bers of B1a cells (3-fold reduction) and B1b cells (2- to

4-fold reduction) in both C57BL ⁄ 6J and BALB ⁄ cByJ mice

(Figure 3c). To investigate whether free, PC B1 cells migrate

into the pristine-induced granulomas, WMT cells were

collected and labelled with antibodies specific for B1 cell

surface markers, B220, CD5, IgM, and CD11b in prepara-

tion for flow cytometric analysis. There were virtually no B1

cells in the mesentery of naı̈ve C57BL ⁄ 6J or BALB ⁄ cByJ

mice (data not shown). Their representation among WMT

cells was <0.3% in both C57BL ⁄ 6J and BALB ⁄ cByJ mice at

weeks 3 and 11, indicating that there were few, if any, B1

cells in the granulomas. Therefore, these free B1 B cells may

undergo apoptosis or leave the PC.

Inflammation mobilises lymphocytes, including immature

B cells from the bone marrow (BM) to blood and the spleen

(Ueda et al. 2004). To determine if immature, CD93+ B cell

immigrants were present in MG and SG, we analysed the

phenotypes of the B cells present in WMT by FACS. Virtu-

ally all B cells in both C57BL ⁄ 6J and BALB ⁄ cByJ WMT at

3 and 11 weeks after pristane injection expressed high levels

of B220 and IgD but no CD93, a surface phenotype indica-

tive of full developmental maturity (Figure 4a). Presence of

mature conventional B cells in the granuloma suggests their

potential role in pristane-induced inflammation.

Plasma cells are recruited to mesenteric oil granulomas

where pristane-induced PCT form when BALB ⁄ cAn mice are

housed conventionally and exposed to incidental infection

and antigens (Potter & Maccardle 1964). To determine

whether plasma cells were also present in the pristine-

induced granulomas in the C57BL ⁄ 6J and BALB ⁄ cByJ mice

maintained under specific-pathogen free conditions, WMT

cells were isolated from these two strains given pristane 3 or

11 weeks earlier, and stained with PE-conjugated CD138

antibody for flow cytometry analysis. CD138 is commonly

used as a marker to identify plasma cells (Rawstron et al.

1997). Interestingly, CD138+ cells were observed exclusively

in the WMT and PC of BALB ⁄ cByJ at 11 weeks after pris-

tane injection (data not shown). The presence of CD138+

cells in the BALB ⁄ cByJ WMT was confirmed in situ by

immunofluorescence and once again, CD138+ cells were only

present in BALB ⁄ cByJ MG sections at week 11 after pristane

(Figure 4b). This finding correlates strikingly with the sus-

ceptibility of BALB ⁄ cByJ and resistance of C57BL ⁄ 6J mice

to pristane-induced PCT development (Potter 2003).

Organisational differences between MG and SG

BALB ⁄ cByJ mice developed lower numbers of SG at week 3

after pristane than did C57BL ⁄ 6J mice (Figure 1b). These

SG were absent in BALB ⁄ cByJ mice by week 11, while they

Strain-specific granuloma responses 465

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

persisted in C57BL ⁄ 6J mice. On the other hand, BALB ⁄ cByJ

MG became increasingly more cellular from weeks 3 to 11,

but became less cellular in C57BL ⁄ 6J mice at week 11 com-

pared to week 3, suggesting that the granuloma response

was resolving in C57BL ⁄ 6J mice (Figures 1a and S1).

Indeed, the cellularity of MG increased in BALB ⁄ cByJ mice

from week 3 to week 11 after pristane, whereas it decreased

in C57BL ⁄ 6J mice (Figure 2c).

What accounts for these differences? One possibility is a

differential distribution pattern of pristane in C57BL ⁄ 6J and

BALB ⁄ cByJ mice. Since pristane droplets have been shown

to be surrounded by phagocytes (Potter & Maccardle 1964),

we labelled phagocytes with India ink by injecting it intra-

peritoneally into mice at week 3 after pristane (Figure 5a).

This allowed us to trace the kinetics of accumulation of pris-

tane-phagocyte aggregates on the mesentery during later

development of granuloma from 3 weeks to 11 weeks, and

provides us with a tool to compare developmental kinetics

of granuloma development in C57BL ⁄ 6J and BALB ⁄ cByJ

mice. Four hours after India ink injection, carbon particles

were distributed evenly in both the MG and SG of BALB ⁄cByJ mice, but were preferentially located in the SG of

C57BL ⁄ 6J mice (data not shown). The differences in India

ink accumulation became more evident at day 12 after ink

injection (Figure 5b) and were dependent on pristane-

induced inflammation, as the patterns of ink distribution in

naı̈ve BALB ⁄ cByJ and C57BL ⁄ 6J mice were similar. Histo-

logically, carbon particles were absorbed on, or internalised

(a) Neutrophils Mφ

60

80

*40

60

**

Total populations05

)

200

300 ***C57BL/6JBALB/cByJ

B cellsDCs12

*6

0

20

40

0 2 4 6 8 10120

20

40

0 2 4 6 8 1012

T cells

erit

on

eal c

avit

y (×

10

0

100

200

0 2 4 6 8 10 12

20**

0

4

8

0 2 4 6 8 1012

* *

0

2

4

0 2 4 6 8 10 12

*

After pristane (weeks)

Cel

ls in

pe

0

5

10

15

0 2 4 6 8 1012

**

p ( )

102

103

104

60.425.3

MD11

b

102

103

104

16.8PI

102

103

104

33.0

B1a B cell CD5–IgM+

B1b B cell CD5+IgM+

al

2B1a B1b

2 C57BL/6J

100 101 102 103 104100

101

CD5

IgM

B220

CD

100 101 102 103 104100

101

FSC-A0 200 400 600 800 1000

100

101

Cel

ls in

per

ito

ne a

cavi

ty (

×105

)

Naïve 3 weeks0

0.5

1

1.5

2

Naïve 3 weeks0

0.5

1

1.5

2 C57BL/6JBALB/cByJ

(b)

(c)

Figure 3 Influx of inflammatory leuco-cytes into the PC following pristaneinjection. (a) Peritoneal cells were har-vested from naı̈ve or pristane injectedC57BL ⁄ 6J (black diamonds) andBALB ⁄ cByJ mice (white diamonds) at3 weeks or 11 weeks. Numbers of totalcells or leucocytes were isolated fromperitoneal lavage, stained and analysedby flow cytometry. (b) In a separatestudy, peritoneal cells harvested at week3 were labelled with B220-PE-Cy7,CD11b-APC-Cy7, CD5-PE and IgM-TxR to identify B1a cells (B220low-

CD5+IgMhiCD11blow) and B1b cells(B220lowCD5-IgMhiCD11blow). (c) Thenumbers of B1a and B1b in the PC areshown. Asterisks indicate significant dif-ferences between C57BL ⁄ 6J andBALB ⁄ cByJ mice: *P < 0.05;**P < 0.01.

466 H. Chen et al.

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

into, CD11b+ cells (Figure 5c). In C57BL ⁄ 6J mice, more

CD11b+ cells were associated with carbon particles in SG

than MG (Figure 5c). In contrast, the difference in distri-

bution of CD11b+ cells containing carbon particles bet-

ween SG and MG was not apparent in BALB ⁄ cByJ mice

(Figure 5c). These data suggest that from week 3 after pris-

tane, peritoneal inflammatory cells were preferentially

recruited to and accumulated in the SG in C57BL ⁄ 6J mice.

This may explain why C57BL ⁄ 6J mice preferentially main-

tained the SG structure and resolved MG. BALB ⁄ cByJ mice

did not demonstrate any such preference. Continuous cell

recruitment promoted the MG development in BALB ⁄ cByJ

mice. SG eventually resolved, though they were still able to

recruit cells, suggesting that other factors might affect the

nodular structure of SG.

Leucocyte recruitment is regulated by their specific chemo-

kines during inflammation (Douglas et al. 2002). M/s are

major CD11b+ expressing phagocytes in pristane-induced

granulomas. To understand the preferential recruitment of

M/ to the mesentery in C57BL ⁄ 6J, but not in BALB ⁄ cByJ

mice, we analysed the expression of the M/ chemokine

MCP-1 by SG cells and MG cells at week 3 after pristane.

Interestingly, C57BL ⁄ 6J SG cells expressed more MCP-1

mRNA than C57BL ⁄ 6J MG cells (Figure 5d). This may

explain why from week 3 after pristane, M/ were preferen-

tially recruited to SG (Figure 5b), therefore leading to a

greater amount of pristane brought to SGs through aggre-

gates of pristane and M/ or other leucocytes. However, in

BALB ⁄ cByJ mice, there was no difference in MCP-1 expres-

sion between the two structures. This result suggests that

from week 3, both SG and MG in BALB ⁄ cByJ mice were

able to recruit M/ or other inflammatory cells, correlating

with the observation that MG and SG recruited CD11b+

cells equally (Figure 5b,c).

Collectively, the differential kinetics of MG development

and resolution in BALB ⁄ cByJ and C57BL ⁄ 6J mice, as well

as the extended duration of SGs in B ⁄ 6 mice, may be due to

differences in the pattern of leucocyte recruitment that is

directed by specific chemokines, such as MCP-1.

Discussion

Granulomateous inflammation acts as a protective response

by walling off persistent living pathogens (Williams & Wil-

liams 1983; Co et al. 2004). However, granulomas induced

by non-infectious agents such as suture material or pristane,

cause immunopathology without any clear benefit to the

host (Williams & Williams 1983; Tang & Eaton 1995). The

present study applied a non-infectious model of the granu-

loma response by injecting pristane into the PC of mice.

Whereas this model was developed almost 50 years ago

(Potter & Maccardle 1964), for the first time we have

(a)

0.001

0.03

0.02

4.0

Naïve 3 weeks

C57BL/6J

BALB/cByJ

CD

93B220 IgD

0

0.06

0.08

7.1

Eve

nts

0.04

3.1

0.1

2.5

11 weeks

MG SG MG

20 μm

C57BL/6J BALB/cByJ

23.3

7.9

Bone marrow

WMT

C57BL/6J

(b)

Figure 4 Characterisation of B cells inoil granulomas. (a) B cells in oil granu-lomas exhibit mature phenotypes(B220hiCD93-). WMT cells were recov-ered from naı̈ve mice or mice injectedwith 300 ll pristane for 3 or 11 weeks,labeled with B220-PE-Cy7, CD93-APCand IgD-PE, and analysed by flowcytometry. BM cells from naı̈veC57BL ⁄ 6J mice were harvested andlabeled as indicative for mature con-ventional B cells (B220hiCD93)IgD+) orimmature B cells (B220lowCD93+IgD)).(b) Plasma cells were only present inBALB ⁄ cByJ MG at week 11. A piece ofMG, or a peripheral strip of mesenterycontaining SG, was excised from pris-tane-treated C57BL ⁄ 6J and ⁄ or BALB ⁄cByJ mice (week 11), and embedded inOCT compound. Sections of 5-lm werecut, and fixed with acetone ⁄ methanol(1:1). Sections were stained withCD138-PE antibody, and visualised byfluorescent microscopy. Original magni-fication (objective): ·20. Arrows indi-cate positive staining.

Strain-specific granuloma responses 467

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

detailed and compared the development of pristane granulo-

mas in two mouse strains, BALB ⁄ cByJ and C57BL ⁄ 6J, that

support, or are resistant to inflammatory PCT (Potter 2003).

In both strains, pristane induced recruitments of inflamma-

tory leucocytes (Figure 2), resulting in the development of

oil granulomas, that could be further divided into MG and

SG (Figure 1a). Both MG and SG contained leucocyte popu-

lations, that were dominated by MF (approximately 20% of

all the leucocytes), followed by T cells and neutrophils

(approximately 15%, each), DCs (approximately 10%) and

conventional B cells (approximately 3%). The cellular com-

position of pristane MG and SG is similar to that of tuber-

culous granulomas (Tsai et al. 2006).

General histological features were also similar between

MG and SG in both strains. Like other causative agents in

granuloma formation (Birkness et al. 2007), pristane was

buried in the mesentery, surrounded by inflammatory leuco-

cytes (Figure 1c). Pristane-induced granulomas had marked

differences in organisation when compared to infectious

granulomas. For example, during infection by tubercle

bacillus, the invading pathogens are first surrounded by

activated M/; this complex is encompassed by lymphocytes.

Pristane granulomas, however, result from pristane being

randomly surrounded by various types of cell populations,

including M/, neutrophils and lymphocytes (Figure 1c). This

difference might be due to, at least in part, differences

between the nature of pristane and living pathogens. Deposi-

tion of collagen and fibrosis in pristane granulomas, com-

monly described in granuloma responses to other stimuli

(Bowers et al. 1980; Restrepo et al. 2001), was confirmed

histologically (Figure 1c).

The pattern of pristane-induced SG and MG development

was quite distinct in the BALB ⁄ cByJ and C57BL ⁄ 6J strains.

BALB ⁄ cByJ mice in this study exhibited similar mesenteric

responses to pristane as those previously described by Potter

and Maccardle (1964), including the development of mesen-

teric polyps, the presence of M/, neutrophils and lympho-

cytes, plasma cells, and the appearance of rare oil

granuloma nodules, or SG, along the junction of mesentery

and gut. Briefly, in response to pristane, MG was the main

PristaneNaïve(b)(a)

C57

BL

/6J

yJ

Pristane India ink sacrifice

0

Week 3

Week 3

BA

LB

/cB

y

SG MG (d)(c)

+day 12

l of

MC

P-1

(re

lati

ve

2

3

4* SG

MG

C57

BL

/6J

yJ

mR

NA

leve

lto

ββ–a

ctin

)

BALB/cByJC57BL/6J0

1

BA

LB

/cB

y

Figure 5 India ink experiments. (a) C57BL ⁄ 6J or BALB ⁄ cByJ mice were injected i.p. with 300 ll pristane, and 3 weeks later wereinjected i.p. with 0.5 ml of a 1 ⁄ 10 dilution of India ink in PBS. Twelve days after ink injection, the PC of mice was opened and gut-associated WMT was isolated and macro-photographed. Mice that were treated with only India ink were included as controls. (b) Arepresentative picture of gut-associated WMT is shown. (c) Immunohistology of MG and SG. Sections were made from a block ofperipheral mesentery containing SGs or an equivalent area of MG and were stained with biotin labelled CD11b, followed by incuba-tion with streptavidin conjugated HRP. A piece of peripheral mesentery corresponding to SG (cSG) or a corresponding area of MG(cMG) in naı̈ve mice was included as control. Original magnification (objective): ·20. (c) mRNA level of MCP-1 was determined inSG (Black columns) and MG (White columns) from C57BL ⁄ 6J or BALB ⁄ cByJ mice. Asterisks indicate significant differences betweenSG and MG: *P < 0.05; **P < 0.01.

468 H. Chen et al.

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

form of oil granuloma in BALB ⁄ cByJ mice; whereas in

C57BL ⁄ 6J mice, SG was the typical form (Figure 1a).

Why does the granuloma response of BALB ⁄ cByJ mice

preferentially take the form of MG, whereas the C57BL ⁄ 6J

mice response to pristane is dominated by SG? MG and SG

contained common leucocytes (Figure 2c,d), and both began

as pristane-associating leucocytes attached to mesenteric

membranes. Injection of India ink to the pristane-treated

mice revealed that phagocytes were diffusely located both at

the SG and MG in BALB ⁄ cByJ mice, but phagocytes were

mainly localised to the SG in C57BL ⁄ 6J mice (Figure 5).

These differences in the distribution of phagocytes became

more prominent by day 12 after injection. This pattern

was not apparent in naı̈ve mice, and we conclude that

inflammation induces the strain-specific patterns of pristane

granulomas.

MCP-1 expression has been demonstrated to promote M/accumulation in granulomas in response to S. mansoni egg

antigens (Chiu & Chensue 2002). To understand the reasons

for the distinct pattern of granuloma responses in BALB ⁄cByJ and C57BL ⁄ 6J mice, we isolated MG and the rare SG

of BALB ⁄ cByJ mice, and SG and the occasional MG of

C57BL ⁄ 6J mice 3 weeks after pristane. In BALB ⁄ cByJ mice,

expression of messenger RNA of MCP-1 in MG was compa-

rable to that in SG (Figure 5d), suggesting that M/-rich

granulomas could develop either in the mesenteric margins

or in mesenteric centres. In contrast, in C57BL ⁄ 6J mice,

MCP-1 messenger RNA expression level was 3-fold higher

in SG than in MG (Figure 5d), suggesting that granuloma

development is preferred at mesenteric margins. This bias

may explain the dominance of SG in C57BL ⁄ 6J mice (Fig-

ure 1a). Collectively, these data suggest that genes con-

trolling expression of inflammatory chemokines affect

the leucocyte recruitment and ultimately the pattern of the

granuloma response to pristane.

Like inflammatory chemokines, inflammatory cytokines

may also affect patterns of granuloma response to pristane.

In the granuloma response to M. tuberculosis, the absence

of tumor necrosis factor a (TNFa) results in poor granu-

loma formation and widespread dissemination of mycobac-

teria (Bean et al. 1999). We measured the expression of

TNFa and IL-6 in the WMT (containing both MG and

SG) from both BALB ⁄ cByJ and C57BL ⁄ 6J mice at week 3

after pristane. TNFa messenger RNA expression in the

WMT in C57BL ⁄ 6J mice was 2-fold higher than that in

BALB ⁄ cByJ mice (Figure S4). Not all inflammatory cyto-

kines were higher in C57BL ⁄ 6J than in BALB ⁄ cByJ mice.

For example, comparable levels of IL-6 messenger RNA

were detected in the WMT of both strains (Figure S4).

These data suggest distinct roles for these cytokines in the

granuloma response to pristane. These roles could be

examined in the future by using mice deficient for each of

these cytokines.

Additionally, other factors may affect granuloma develop-

ment. For example, circulating leucocytes contribute to oil

granuloma formation (unpublished data). The expression of

adhesion molecules in the mesentery and the pool of circu-

lating leucocytes may therefore have a role in regulating oil

granuloma formation. Also, gender-specific differences in

granuloma responses to pristane were obvious. Male

C57BL ⁄ 6J mice developed a less severe granuloma response

than age-matched female C57BL ⁄ 6J mice, as evidenced by

less cellular MG and fewer SG (Figure S2a,b). This was

confirmed by the fact that the frequency of cells in male

C57BL ⁄ 6J MG was <20% of that in female C57BL ⁄ 6J MG

(Figures S2c and S3a). There was no difference in the

frequency of cells in SG between the two genders (Figure S2d

and S3b). The cellularity of WMT in male C57BL ⁄ 6J mice

was <25% of that in female counterparts (Figures S2e and

S3c). Recruitment of leucocytes to the WMT in male

C57BL ⁄ 6J mice was not as efficient as in age-matched

female mice (Figure S3). The polyps that appeared on the

male C57BL ⁄ 6J mesentery were not observed in female

C57BL ⁄ 6J mice (Figure S2a). In the case of granulomas

induced by infectious pathogens, inefficient sequestration of

these microorganisms may permit higher incidence of escape,

resulting in the progression of disease. A sexual bias is also

evident in the epidemiological patterns of tuberculosis,

where 70% more males than females receive smear-positive

tuberculosis notifications (Diwan & Thorson 1999). Sex-

based differences in pristane responses may reflect potential

roles for sex-associated factors, including hormones (for

example, oestrogen), or genes on the Y chromosome, in oil

granuloma development.

The pristane-induced granuloma represents a non-infec-

tious granuloma, and this study may provide implications

for the granulomateous pathology induced by implantation

of inert surgical biomaterials (Zeller 1983; Tang & Eaton

1995; Griffiths et al. 1996; Nicolau 2007). Dissecting the

regulatory pathways and genes of granuloma responses to

pristane may lead to a better understanding of this impor-

tant mechanism of immunity.

Acknowledgements

We thank Dr. Michael Potter (Laboratory of Genetics,

National Cancer Institute, Bethesda, Maryland) for valuable

discussions and advice and for his critical review of the man-

uscript. We thank Pilar Snowden and Kathleen O’Hara for

assistance in preparing the manuscript. This work was sup-

ported in part by NIH grants AI056363 and AI024335.

Disclosures

The authors declared they have neither financial nor per-

sonal conflict of interest.

References

Bean A.G., Roach D.R., Briscoe H. et al. (1999) Structural deficien-

cies in granuloma formation in TNF gene-targeted mice underlie

the heightened susceptibility to aerosol Mycobacterium tuberculo-

sis infection, which is not compensated for by lymphotoxin.

J. Immunol. 162, 3504–3511.

Strain-specific granuloma responses 469

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

Birkness K.A., Guarner J., Sable S.B. et al. (2007) An in vitro model

of the leukocyte interactions associated with granuloma formation

in Mycobacterium tuberculosis infection. Immunol. Cell Biol. 85,

160–168.

Bowers R.R., Houston F., Clinton R., Lewis M., Ballard R. (1980)

A histological study of the carrageenan-induced granuloma in the

rat lung. J. Pathol. 132, 243–253.

Chiu B.C. & Chensue S.W. (2002) Chemokine responses in schisto-

somal antigen-elicited granuloma formation. Parasite Immunol.

24, 285–294.

Co D.O., Hogan L.H., Il-Kim S., Sandor M. (2004) T cell contribu-

tions to the different phases of granuloma formation. Immunol.

Lett. 92, 135–142.

Diwan V.K. & Thorson A. (1999) Sex, gender, and tuberculosis.

Lancet 353, 1000–1001.

Douglas M.R., Morrison K.E., Salmon M., Buckley C.D. (2002)

Why does inflammation persist: a dominant role for the stromal

microenvironment? Expert Rev. Mol. Med. 2002, 1–18.

Ginsberg J.E. & Becker L.A. (1951) Silicon granuloma of skin due

to traumatic sand inoculation. J. Am. Med. Assoc. 147, 751–753.

Griffiths M.M., Langone J.J., Lightfoote M.M. (1996) Biomaterials

and Granulomas. Methods 9, 295–304.

Hauser F.V. & Rothman S. (1950) Monilial granuloma; report of

a case and review of the literature. Arch. Derm. Syphilol. 61,

297–310.

Kido M., Kajiki A., Nagata N., Manabe H., Iwata Y. (1995) A case

of pulmonary hyalinizing granuloma with its occupational history

of dust exposure. J. Uoeh. 17, 31–37.

Lattanzio G., Libert C., Aquilina M. et al. (1997) Defective develop-

ment of pristane-oil-induced plasmacytomas in interleukin-6-defi-

cient BALB ⁄ c mice. Am. J. Pathol. 151, 689–696.

Ley A., Jacas R., Oliveras C. (1951) Torula granuloma of the cervi-

cal spinal cord. J. Neurosurg. 8, 327–335.

Mantovani A., Sozzani S., Bottazzi B. et al. (1993) Monocyte che-

motactic protein-1 (MCP-1): signal transduction and involvement

in the regulation of macrophage traffic in normal and neoplastic

tissues. Adv. Exp. Med. Biol. 351, 47–54.

Meyer T.J., Ribi E., Azuma I. (1975) Biologically active components

from mycobacterial cell walls. V. Granuloma formation in mouse

lungs and guinea pig skin. Cell. Immunol. 16, 11–24.

Nacionales D.C., Kelly K.M., Lee P.Y. et al. (2006) Type I inter-

feron production by tertiary lymphoid tissue developing in

response to 2,6,10,14-tetramethyl-pentadecane (pristane). Am. J.

Pathol. 168, 1227–1240.

Nicolau P.J. (2007) Long-lasting and permanent fillers: biomaterial

influence over host tissue response. Plast. Reconstr. Surg. 119,

2271–2286.

Nordan R.P., Mock B.A., Neckers L.M., Rudikoff S. (1989) The

role of plasmacytoma growth factor in the in vitro responses of

murine plasmacytoma cells. Ann. N Y Acad. Sci. 557, 200–205.

Potter M. (2003) Neoplastic development in plasma cells. Immunol.

Rev. 194, 177–195.

Potter M. & Maccardle R.C. (1964) Histology of developing plasma

cell neoplasia induced by mineral oil in Balb ⁄ C mice. J. Natl Can-

cer Inst. 33, 497–515.

Potter M., Pumphrey J.G., Walters J.L. (1972) Growth of primary

plasmacytomas in the mineral oil-conditioned peritoneal environ-

ment. J. Natl Cancer Inst. 49, 305–308.

Potter M., Mushinski E.B., Wax J.S., Hartley J., Mock B.A. (1994)

Identification of two genes on chromosome 4 that determine

resistance to plasmacytoma induction in mice. Cancer Res. 54,

969–975.

Rawstron A.C., Owen R.G., Davies F.E. et al. (1997) Circulating

plasma cells in multiple myeloma: characterisation and correlation

with disease stage. Br. J. Haematol. 97, 46–55.

Restrepo B.I., Alvarez J.I., Castano J.A. et al. (2001) Brain granulo-

mas in neurocysticercosis patients are associated with a Th1 and

Th2 profile. Infect. Immun. 69, 4554–4560.

Silva S., Kovalchuk A.L., Kim J.S., Klein G., Janz S. (2003) BCL2

accelerates inflammation-induced BALB ⁄ c plasmacytomas and

promotes novel tumors with coexisting T(12;15) and T(6;15)

translocations. Cancer Res. 63, 8656–8663.

Tang L. & Eaton J.W. (1995) Inflammatory responses to biomaterials.

Am. J. Clin. Pathol. 103, 466–471.

Tsai M.C., Chakravarty S., Zhu G. et al. (2006) Characterisation of

the tuberculous granuloma in murine and human lungs: cellular

composition and relative tissue oxygen tension. Cell Microbiol. 8,

218–232.

Ueda Y., Yang K., Foster S.J., Kondo M., Kelsoe G. (2004) Inflam-

mation controls B lymphopoiesis by regulating chemokine

CXCL12 expression. J. Exp. Med. 199, 47–58.

Warren K.S. & Domingo E.O. (1970) Granuloma formation around

Schistosoma mansoni, S. HAEMATOBIUM, AND S. Japonicum

eggs. Size and rate of development, cellular composition, cross-

sensitivity, and rate of egg destruction. Am. J. Trop. Med. Hyg.

19, 292–304.

Williams G.T. & Williams W.J. (1983) Granulomatous inflamma-

tion – a review. J. Clin. Pathol. 36, 723–733.

Wooley P.H., Seibold J.R., Whalen J.D., Chapdelaine J.M. (1989)

Pristane-induced arthritis. The immunologic and genetic features

of an experimental murine model of autoimmune disease. Arthritis

Rheum. 32, 1022–1030.

Zeller J.M. (1983) Surgical implants. Physiological response. AORN

J. 37, 1284–1291.

Supporting information

Additional Supporting Information may be found in the

online version of this article:

Figure S1 Dose dependent response of mesentery to

pristane. BL ⁄ 6 mice were i.p. injected with 100 or 300 ll

pristane and were harvested at several intervals thereafter.

At each time point, three or more mice were killed and

gut-associated with mesentery was macrophotographed.

Representative photos were shown here. Untreated mice

were used as controls.

Figure S2 Less responses of male BL ⁄ 6 mice to pristane

than female BL ⁄ 6 mice. A: Male BL ⁄ 6 (mBL ⁄ 6) mice were

given 300 ll pristane intraperitoneally and sacrificed at

3 weeks post pristane. Representative macrophotographs of

gut-associated whole mesenteric tissue (WMT) from naive

and pristane-administrated male BL ⁄ 6 mice are shown. B:

Number of serosal granulomas (SGs) developed in male mice

were numerated, and number of SGs in female B ⁄ L6 (fBL ⁄ 6)

was included as a comparison. C: Fewer cells in male OG

than in female OG. D: Frequency of cells in MG. It is

shown as the number of cells in every 1 mm2 of mesentery.

E: Frequency of cells in SG developed at week 3 after pris-

tane, which was expressed as the number of cells per 1 indi-

vidual SG. Values represent the mean and standard error of

three or more mice per group. 6–8 individual SG from 3

470 H. Chen et al.

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471

mice were pooled together, which gave rise to the average

and SD. Asterisks indicate significant differences between

female (black columns) or male BL ⁄ 6 mice (white columns):

*, P < 0.05; **, P < 0.01; ***, P < 0.001

Figure S3 Cellularity of WMT, MG and SG in male and

female BL ⁄ 6 mice. A: Frequency of cells in MG. MG cells

were collected from mice treated with pristane for 3 weeks,

and were labeled with antibodies as described in material

and methods. B: Frequency of cells in SG. 6-8 individual

SGs were isolated from 3 mice at week 3 after pristane,

digested with collagenases to release cells. C: Cellularity of

WMT. WMT cells were harvested from naı̈ve mice or mice

injected with pristane for 3 weeks, and labeled with antibod-

ies and analyzed as described in Material and Methods.

Asterisks indicate significant differences between mBL ⁄ 6 and

fBL ⁄ 6 mice: *, P < 0.05; **, P < 0.01, ***, P < 0.001.

Figure S4 Expression of cytokines by WMT cells. WMT

cells were isolated from BL ⁄ 6 (filled columns) and B ⁄ c (empty

columns) mice treated without or with 300 ll pristane for

3 weeks. mRNA from these cells was quantified by quantative

PCR with specific primers. Asterisks indicate significant dif-

ferences between BL ⁄ 6 and B ⁄ c mice: **, P < 0.01.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials supplied

by the authors. Any queries (other than missing material)

should be directed to the corresponding author for the article.

Strain-specific granuloma responses 471

� 2010 The Authors. Journal compilation � 2010 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 91, 460–471