MIF Receptor CD74 is Restricted to Microglia/Macrophages, Associated with a M1-Polarized Immune Milieu and Prolonged Patient Survival in Gliomas - PubMed (original) (raw)

. 2015 Jul;25(4):491-504.

doi: 10.1111/bpa.12194. Epub 2014 Nov 20.

Corinna Preusse 2, Anna-Eva Blank 1, Cornelia Zachskorn 1 3 4, Peter Baumgarten 1 5, Lixi Caspary 1, Anne K Braczynski 1, Jakob Weissenberger 6, Hansjürgen Bratzke 7, Sandy Reiß 8, Sandra Pennartz 8, Ria Winkelmann 9, Christian Senft 6 5 3, Karl H Plate 1 3 4, Jörg Wischhusen [ 10](#full-view-affiliation-10 "Junior Research Group "Tumour Progression and Immune Escape", Interdisciplinary Center for Clinical Research, Department for Obstetrics and Gynecology, University of Würzburg, Würzburg, Germany."), Werner Stenzel 2, Patrick N Harter 1 3 4, Michel Mittelbronn 1 3 4

Affiliations

MIF Receptor CD74 is Restricted to Microglia/Macrophages, Associated with a M1-Polarized Immune Milieu and Prolonged Patient Survival in Gliomas

Pia S Zeiner et al. Brain Pathol. 2015 Jul.

Abstract

The macrophage migration inhibitory factor (MIF) receptor CD74 is overexpressed in various neoplasms, mainly in hematologic tumors, and currently investigated in clinical studies. CD74 is quickly internalized and recycles after antibody binding, therefore it constitutes an attractive target for antibody-based treatment strategies. CD74 has been further described as one of the most up-regulated molecules in human glioblastomas. To assess the potential relevance for anti-CD74 treatment, we determined the cellular source and clinicopathologic relevance of CD74 expression in human gliomas by immunohistochemistry, immunofluorescence, immunoblotting, cell sorting analysis and quantitative polymerase chain reaction (qPCR). Furthermore, we fractionated glioblastoma cells and glioma-associated microglia/macrophages (GAMs) from primary tumors and compared CD74 expression in cellular fractions with whole tumor lysates. Our results show that CD74 is restricted to GAMs in vivo, while being absent in tumor cells, the latter strongly expressing its ligand MIF. Most interestingly, a higher amount of CD74-positive GAMs was associated with beneficial patient survival constituting an independent prognostic parameter and with an anti-tumoral M1 polarization. In summary, CD74 expression in human gliomas is restricted to GAMs and positively associated with patient survival. In conclusion, CD74 represents a positive prognostic marker most probably because of its association with an M1-polarized immune milieu in high-grade gliomas.

Keywords: CD74; MIF; glioma; immune polarization.

© 2014 International Society of Neuropathology.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

CD

74

mRNA

levels increase with the grade of malignancy in human glioma tissue. The

R

epository of

M

olecular

B

rain

N

eoplasia

D

ata (

REMBRANDT

) data analysis of the

CD

74 gene expression (median expression × 103) in 454 gliomas [

REMBRANDT N

ational

I

nstitutes of

H

ealth (

NIH

) 2011;

https://caintegrator.nci.nih.gov/rembrandt/

]. Three different probesets were studied: 1567627_at (red; on chromosome 5 at 149.762876 Mb on the plus strand); 1567628_at (blue; on chromosome 5 at 149.762876 on the minus strand); 209619_at (green; on chromosome at 149.761492 Mb on the minus strand).

Figure 2

Figure 2

CD

74 expression is absent in human malignant glioma cell lines. In double‐immunofluorescence (

IF

) staining

CD

74 (

A

lexa 568, red) was absent in all tested glioma cell lines (not shown), only glial fiber acid protein (

GFAP

) (

A

lexa 488, green) was positive in (A)

U

251 and (B)

U

373 (original magnification ×10 in A and B; images taken with the

E

clipse 80

i

fluorescent microscope). While human lymphoma cell lines

R

amos (C) and

R

aji (D) exhibited

CD

74 expression on the cell surface thereby serving as positive controls, glioma cell lines (E–K) did not show

CD

74 expression. The

CD

74 immunoblot from glioma cell lysates and human tonsil as positive control showed a complete absence of

CD

74 protein in glioma cell lines (L).

Figure 3

Figure 3

CD

74 is strongly expressed in normal and neoplastic central nervous system (

CNS

) tissue in vivo. Representative immunhistochemical stainings showing

CD

74 expression on a distinct cellular subset in (A) normal‐appearing grey matter (n = 44), (B) normal‐appearing white matter (n = 16) and astrocytomas of (C)

W

orld

H

ealth

O

rganization (

WHO

) grade

I

(n = 47), (D)

WHO

grade

II

(n = 16), (E)

WHO

grade

III

(n = 35) as well as (F) glioblastoma,

WHO

grade

IV

(n = 252). Histomorphologic characteristics of

CD

74‐positive cells were also analyzed with regard to the microlocalisation such as (F)

GBM

tumor centers (n = 252), (G)

GBM

infiltration zones (n = 37) or (H) in

GBM

recurrence (n = 114). While in tumor centers of both primary (F) and recurrent (H)

GBMs CD

74 expression was detected on the surface of mostly rounded to ovoid cells,

CD

74 expression in normal

CNS

tissue (A, B) and

GBM

infiltration zones (G) was mainly observed on elongated cellular protrusions. (scale bars = 50 μm; inserts = higher magnification).

Figure 4

Figure 4

CD

74 expression is differentially regulated in astrocytic tumors and normal central nervous system (

CNS

) tissues.

B

ox and

W

hisker plots for

CD

74‐positive cells (in %) in astrocytomas

I

–

IV

,

GBM

infiltration zones as well as normal‐appearing

CNS

tissues are depicted. The relative amount of

CD

74‐positive cells in pilocytic astrocytoma,

W

orld

H

ealth

O

rganization (

WHO

) grade

I

(n = 47); diffuse astrocytoma

WHO

grade

II

(n = 16); anaplastic astrocytoma

WHO

grade

III

(n = 35); glioblastoma

WHO

grade

IV

(n = 252),

GBM

infiltration zone (n = 37), normal‐appearing white matter of

GBM

samples (

NAWM

; n = 16) as well as normal‐appearing grey matter of

GBM

samples (

NAGM

; n = 44) was statistically assessed using the nonparametric

W

ilcoxon's test. A significance level of alpha = 0.05 was chosen for all testings. For adjustment of the _P_‐values because of multiple testing we used the method of

B

onferroni–

H

olm. Statistical analysis was performed using

JMP

8.0.1 software (

SAS

).

Figure 5

Figure 5

CD

74 expression in vivo is restricted to microglia cells in normal central nervous system (

CNS

) tissues as well as glioma‐associated microglia/macrophages (

GAMs

) in gliomas. Representative double‐immunofluorescence (

IF

) stainings of

CD

74 (

A

lexa 488, green) and the microglia/macrophage marker Iba1 (

A

lexa 568, red) show a colocalization in (A) normal white (n = 4), (B) grey matter (n = 4) and (C) glioblastoma World Health Organization (

WHO

) grade

IV

(n = 15) samples (images A–C were taken with the

C

1 confocal microscope). No colocalization of the ligand migration inhibitory factor (

MIF

) (

A

lexa 488, green) and its receptor

CD

74 (

A

lexa 568, red) was seen in (D) normal brain or (E) glioblastoma

WHO

grade

IV

samples (images D–E were taken with the

E

clipse 80

i

fluorescent microscope; original magnification A–D: 20×; E: 40×). In addition, pilocytic astrocytoma

WHO

grade

I

(n = 2) as well as

WHO

grade

II

(n = 2) and

III

(n = 2) astrocytomas were assessed revealing similar staining results (data not shown).

Figure 6

Figure 6

CD

74

mRNA

and protein levels correlate with the content of glioma‐associated microglia/macrophages (

GAMs

). Hierarchical cluster analyses (A) of gene expression signatures of selected key factors of immune cells, glioblastoma cells as well as glioblastoma micromilieu of 424 primary glioblastomas and 11 normal brain samples deriving from the cancer genome atlas (

TCGA

) data portal using the

A

gilent 244

K G

4502

A

microarray to determine

mRNA

profiles. Hierarchical clustering of this data was performed using the

W

ard's minimum variance method. Dark red color indicates a perfect positive correlation (+1) gradually decreasing to a perfect negative correlation (−1) indicated in blue. Correlation analyses of the same data subset comparing

CD

74 with either (B) Iba1 (

AIF

1), (C)

CD

68 or (D) CD11

b

(

ITGAM

) are depicted.

CD

74 and Iba1 immunoblotting (E) of

GAMs

, glioblastoma cells (

PC

) and whole glioblastoma tissue (

T

) of three different patients with primary glioblastoma

W

orld

H

ealth

O

rganization (

WHO

) grade

IV

showing a positive correlation of

CD

74 and Iba1 protein expression. Actin served as positive control.

Figure 7

Figure 7

The migration inhibitory factor (

MIF

)‐

CD

74 system is associated with a distinct

M

1‐polarized, phagocytic glioma‐associated microglia/macrophage (

GAM

)‐phenotype in glioblastomas. Light microscopy of unstimulated

GAMs

(A) or

GAMs

after stimulation with (B) recombinant human

MIF

(

rhMIF

) 10 ng/mL, (C) human recombinant macrophage colony stimulating factor (

rhM

‐

CSF

) 10 ng/mL, (D)

rhMIF

10 ng/mL and

rhM

‐

CSF

10 ng/mL, (E)

rhMIF

10 ng/mL and neutralizing anti‐

CD

74 antibody (

GAM

in A–E were derived from glioblastoma

W

orld

H

ealth

O

rganization (

WHO

) grade

IV

). (

F

) Differential mRNA expression for classical M1/M2 molecules as assessed by quantitative polymerase chain reaction (

qPCR

) [using

T

aq

M

an

F

ast

U

niversal

PCR M

aster

M

ix, the target assay (

A

pplied

B

iosystems) and 20 ng of complementary DNA (

cDNA

) ] did not reveal any considerable differences between the stimulation of

GAMs

with

MIF

and an anti‐

CD

74 approach as compared with the

IgG

1 isotype (at the same concentration as anti‐

CD

74 antibody) or negative (bovine serum albumin at 10 ng/mL also serving as diluent for

rhMIF

) control. (G) Analyses of the cancer genome atlas (

TCGA

) data using the

R

2 platform revealed a distinct, mainly

M

1‐based profile with additional phagocytic or adhesion molecules (correlation with

CD

74 expression is given in descending order).

Figure 8

Figure 8

The amount of

CD

74‐positive glioma‐associated microglia/macrophages (

GAMs

) is an independent prognostic marker for patient survival in

GBM

.

K

aplan–

M

eier survival curves of

GBM

patients were obtained by performing median split for

CD

74 (high expression > 15%

CD

74‐positive

GAMs

; low expression ≤ 15%

CD

74‐positive

GAMs

) levels. Curves were compared by both log–rank (P = 0.0018) and

W

ilcoxon (P = 0.0066) tests. Multivariate analysis was performed using the

C

ox proportional hazard model controlling for

CD

74‐positive

GAMs

(positive cells/all cells),

Ki

67 proliferation rate,

CD

68‐positive

GAMs

, sex and patient age.

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