THP-1 macrophages and monocyte-derived macrophages as an alveolar macrophage model

Melanie Erriah1, 2, Kavita Pabreja1, 2, Katherine J Baines1, 2, 3, Michael Fricker1, 2, Jodie L Simpson1, 2

1Priority Research Centre for Healthy Lungs, Faculty of Health and Medicine, University of Newcastle, Callaghan, New South Wales 2300, Australia, 2Hunter Medical Research Institute, University of Newcastle, Callaghan, New South Wales 2300, Australia, 3Department of Respiratory and Sleep Medicine, Hunter

New England Area Health Service, Newcastle, New South Wales 2305, Australia

INTRODUCTION

• Alveolar macrophages can often be difficult to isolate in sufficient numbers and with a good viability. The use of macrophage models such as THP-1 cells and monocyte-derived macrophages eliminates these limitations as it provides an adequate supply of viable cells which can be used to study macrophage biology.

• The THP-1 cell line is generally used as a monocyte model as it has been shown to express a range of markers commonly found on human monocytes (Fig. 1).

• THP-1 cells can be differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA).

Fig. 1 May-Grünwald-Giemsa stained cytospin slides of (A) THP-1 cells and (B) human monocytes (original magnification x 400)

• Monocyte-derived macrophages (MDMs) are primary cells derived from peripheral blood monocytes that have been treated with the growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF).

• CD68 is a well-characterised pan-macrophage marker that is highly expressed intracellularly in human monocytes and macrophages.

• CD98 is a cell surface glycoprotein involved in cell adhesion and amino acid transport expressed on a range of leucocytes including macrophages.

AIM: To compare the morphology and marker expression of these two types of macrophages to determine which one is

best suited as an alveolar macrophage model.

METHODS

• THP-1 cells at different passage numbers (n=7) were stimulated with 100ng/ml PMA for three days, then rested for five days in PMA-free media to produce THP-1 macrophages.

• MDMs were generated by incubating monocytes from healthy volunteers (n=8) for 12 days in the presence of 2ng/ml GM-CSF.

• The cell morphology was assessed by microscopic analysis of May-Grünwald-Giemsa stained cytospin slides.

• The cell viability was assessed by trypan blue exclusion.

• The proportion of cells expressing the macrophage markers CD68 and CD98 was assessed by flow cytometry using CD68-BV421 and CD98-FITC antibodies (BD Biosciences, North Ryde, NSW, Australia).

RESULTS

• THP-1 cells were used between passages 10 and 20 inclusive. The MDM donor characteristics are given in Table 1 below:

Table 1 summarising the MDM donor demographics. Data is given as median (Q1, Q3) unless otherwise indicated.

• THP-1 macrophages and MDMs had a significantly larger cytoplasmic volume than their precursors and had the characteristic “fried egg” appearance of alveolar macrophages (Fig. 2).

Fig. 2 May-Grünwald-Giemsa stained cytospin slides of (A) THP-1 macrophages and (B) monocyte-derived macrophages (original magnification x 400)

• The viability (median (Q1, Q3)) of THP-1 macrophages (89 (82, 93)%) was significantly higher than that of MDMs (63 (59, 79)%) (p = 0.004).

• The intracellular expression of CD68 was significantly higher in MDMs compared with THP-1 macrophages (Fig. 3A). Surface CD98 expression was similar between the two types of macrophages (Fig. 3B).

Fig. 3 Graphs showing the median proportion of macrophages expressing (A) intracellular CD68 and (B) surface CD98.

CONCLUSIONS

• THP-1 macrophages and MDMs exhibited similar morphological traits as alveolar macrophages.

• The lack of the macrophage-specific marker CD68 in THP-1 macrophages may be due to them being an immortalised cell line.

• MDMs being a primary cell, highly expressed both markers, which indicate that they are a more accurate model of airway macrophages.

ACKNOWLEDGEMENTS

This work was supported by an NHMRC project grant and a University of Newcastle International Postgraduate Research Scholarship. Catherine Delahunty, Lorissa Hopkins and Stephany Sanchez for blood collection.

Nicole Cole for flow cytometry.

N 8Age years 31.5 (26.0, 42.8)Sex male, n (%) 4 (50)BMI 22.8 (19.9, 23.3)

THP-1 macrophages MDMs0

50

100

CD

68 e

xpre

ssio

n (%

)

P < 0.001

THP-1 macrophages MDMs0

50

100

CD

98 e

xpre

ssio

n (%

)

P = 0.088

A B

A

A B

B