The liver is populated by a broad spectrum of markers for macrophages. In alcoholic hepatitis the macrophages are M1 and M2

Abstract

Background: Liver cell injury in alcoholic hepatitis (AH) is in part, due to macrophage generated proinflammatory cytokines i.e., M1, M2a, M2b, and M2c might be involved in ALD. The T cell response to chemokines and cytokines differs not only when M1 and M2 macrophages are compared but even when individual M2 subtypes are profiled.

Purpose: In AH, M1 monocytes in the blood show increased sensitivity in the TNF-α response to LPS. Immunohistochemistry (IHC) studies showed that the liver sinusoids in ALD are abundantly populated by CD163 expressing type 2 macrophages. In this report, we profile many of the molecules associated with M1 and M2 macrophages in livers with AH using IHC.

Methods: Using immunofluorescent antibody-labeling, we profiled the receptors, cytokines and chemokines observed in M1, M2a, M2b, and M2c macrophages in liver biopsies from patients with AH.

Results: The increased CD 163 expression found in previous studies was confirmed as well an additional macrophage phenotypic marker CD206, suggesting that AH pathogenesis at least partially involves M2a and M2c macrophages. TGF-β was found to be robustly over expressed by liver sinusoidal macrophages. Macrophage expression of the phenotypic markers TLR-2, TLR-4 and TLR-8 - found in both M1 and M2 macrophages - as well as the chemokines CCL-1 and CCL-18 was found. However, IRF-4, which is related to IL-4 production and M2a polarization as well as the cytokines CCL-1 and Il-1β and the chemokine CXCL-1 were also observed, suggesting that M2a and M2b also play a role in AH pathogenesis.

Conclusion: Livers with AH show robust macrophage over expression of TGF-β, a growth factor more commonly associated with M2 type macrophages and mostly known for its fibrogenetic properties. However, our immunoprofiling of macrophage over expression also shows that AH is driven by receptors, interferons, and cytokines that are commonly associated not just with M2 macrophages, but with M1 as well. Thus, a complex interplay between different types of macrophages expressing a diverse array of molecules and receptors is involved in AH.

Keywords: Alcoholic hepatitis; CD163; Macrophages; TLR-4.

Fig. 1

The antibody to F4/80 showed numerous positive macrophages in liver sinusoids staining green (A) in a patient with alcoholic hepatitis. The tricolor filter (B) showed that macrophages stain strongly positive alongside red blood cells (red colored). Magnification: A, B ×650. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The antibody to CD163 showed numerous positive macrophages stained green (A) in a patient with severe alcoholic hepatitis and with Mallory–Denk body (MDB) formation. Stain for the antibody to ubiquitin stained the MDBs red (B) The tricolor filter showed that the CD163 positive macrophages surrounded the liver cell that formed the Mallory body (C) indicating that the macrophages are responding to the Mallory body affected liver cell. The control showed scattered CD163 positive macrophages (D–F). Magnification: A–C ×650. D–F ×433. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

The antibody to CXCL9 showed numerous positive macrophages stained green (A, arrow) in a patient with alcoholic hepatitis. Stain for the antibodies to CD206 stained the macrophages red (B, arrow). Using the double stain with the tricolor filter showed that CD206 is positive in the macrophages (C, arrow). The control showed that CXCL-9 was not expressed by sinusoidal macrophages (D). The control showed sinusoidal CD206 positive macrophages (E). The control tricolor was negative (F). Magnification: A–F ×433. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

The antibodies to CD14 showed numerous positive macrophages stained green (A, arrow) in a patient with alcoholic hepatitis. The stain for the antibody to TGF-beta stained the macrophages red (B, arrow). Using the double stain with the tricolor filter showed that CD14 and TGF-beta co-localize in the macrophages (C, arrow). The control showed that CD14 (D) and TGF-beta (E) were not expressed by sinusoidal macrophages. The control tricolor was negative (F). In the controls, the only positive staining was for lipofuscin in hepatocytes (D, arrow). Magnification: A–F ×433. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

In livers with AH, both the macrophages (arrows) and the liver parenchymal cells show increased intensity of staining for TLR-4 indicating up regulation of expression (A and C). Tricolor filter (B and E). The control showed low intensity staining in liver cells (D). Magnification: A–C ×433, D and E ×650. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

The antibody to TLR2 showed numerous positive macrophages stained green (A, arrow) in a patient with alcoholic hepatitis. Using the TLR2 stain with the tricolor filter showed that macrophages stained strongly positive (B, arrow). In the control, TLR-2 was not expressed by the sinusoidal macrophages (C). The control tricolor was also negative (D). Magnification: A–D ×217. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

The antibody to TLR8 showed numerous positive macrophages stained green (A–C, arrows) in a patient with alcoholic hepatitis. Figure C shows numerous TLR-8 positive macrophages in an area of steatosis. The control showed that TLR-8 was not expressed by sinusoidal macrophages (D). The control tricolor was negative (E). Magnification: A ×108, B ×217, C ×650, D and E ×433. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

In a liver from a patient with alcoholic hepatitis, TLR4 was over expressed in the hepatocytes. Figure A shows that the macrophage (green arrow) and a hepatocyte (red arrow) are over expressing TLR4. Figure B shows ubiquitin staining the MDB red. Figure C shows co-localization of TLR4 with ubiquitin in the MDB-forming hepatocyte (yellow) which is over expressing TLR-4 (green rim around MDB, arrow). Magnification: A–C ×1083. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

The antibody to IL-1β showed numerous positive macrophages stained green (A, arrow) in a patient with alcoholic hepatitis. Stain for the antibody to IRF4 stained the macrophages red (B). Using the double stain with the tricolor filter showed that IL-1 is co-localized with IRF4 (yellow) in the macrophages (C, arrow). The control showed sinusoidal Il-1 positive macrophages (D). The control showed that IRF4 was not expressed by sinusoidal macrophages (E). The control tricolor was negative (F). Magnification: A–F ×433. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 10

The antibody to CCL-1 showed numerous positive macrophages stained green (A) in a patient with alcoholic hepatitis. Using the TLR2 stain with the tricolor filter showed that macrophages stain strongly positive (B). The control showed that TLR-2 was not expressed by sinusoidal macrophages (C). The control tricolor was negative (D). Magnification: A–D ×433. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)