Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Summary

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Abstract

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNFα) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Introduction

Macrophages are innate immune cells, which play multiple roles in the immune response to infection or injury. Macrophages are responsible for initiating an immune response to infection or tissue damage, stopping the inflammatory response, and promoting the healing response¹. Examples of the three best studied macrophage activation states are: 1) macrophages treated with interferon gamma (IFNγ) and bacterial lipopolysaccharide (LPS), designated M(IFNγ + LPS), which contribute to the inflammatory response; 2) macrophages stimulated with interleukin 4 (IL-4), M(IL-4), which are associated with the healing response; 3) macrophages stimula....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of British Columbia.

1. Bone Marrow Macrophage Derivation and Activation with Antibodies

1. Perform euthanasia using CO₂ asphyxiation.
   1. Place mouse in induction chamber. Euthanize mouse with 5% isoflurane anesthesia, for 60 - 90 s, until immobile and breathing is deep and slow.
   2. Turn off isoflurane anesthesia and administer 6 - 8 L/min of CO₂ until mouse has stopped breathing. Leave mouse with CO₂ on for at least another 5 min. Verify that there is no longer....

Results

Murine bone marrow derived macrophages can be cultured from hematopoietic cell precursors in bone marrow aspirates. Bone marrow aspirates pooled from femurs and tibias of one C57BL/6 mouse typically yield 10⁷ bone marrow derived macrophages, making them a convenient source of macrophages for experiments. BMDMs can be used to test antibody based drug responses when challenged with an inflammatory stimulus in vitro. Figure 1 shows that IVIg .......

Discussion

Macrophage activation states play an important role in both tissue homeostasis and disease²². Macrophages can have distinct as well as overlapping activation states, depending on cues in their microenvironment³. They have distinct roles in all stages of the inflammatory response: defense against pathogens, wound healing and tissue restitution, and also have a distinct anti-inflammatory activation state that is important for turning off the inflammatory response

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Iscove’s modified Dulbecco’s medium (IMDM)	Life technologies	12440053
Fetal Bovine Serum (FBS)	Life technologies	12483-020
Recombinant murine macrophage colony stimulating factor (MCSF)	Stemcell technologies	78059
Penicillin-streptomycin	Life technologies	15140148
Monothioglycerol (MTG)	Sigma	88640
1X red blood cell lysis buffer	eBioscience
Cell dissociation buffer	Life technologies	13150016	Enzyme-Free, Hanks's-based, EDTA
Lipopolysaccharide (LPS)	Sigma aldrich	L 4516	From E. coli 0127:B8
IVIg (Gammunex)	Grifols	Received from BC Children's Hospital, Transfusion Medicine
IVIg (Gammagard liquid)	Baxter Healthcare Corporation	Received from BC Children's Hospital, Transfusion Medicine
IVIg (Octagam)	Octapharma	Received from BC Children's Hospital, Transfusion Medicine
Phosphate buffered saline (PBS) (sterile), pH 7.4	Life technologies	10010023
mouse IL-10 ELISA	BD biosciences	555252
mouse IL-12/23p40 ELISA	BD biosciences	555165
anti-Erk1/2 antibody	Cell signalling technology	9101
anti-pp38 antibody	Cell signalling technology	9211
anti-GAPDH antibody	Fitzgerald industries	10R-G109A
26 g needle	BD biosciences	305110
1 mL syringe	BD biosciences	309659
10 mL syringe	BD biosciences	309604
15 mL conical tube	BD biosciences	352096
50 mL conical tube	BD biosciences	352070
microcentrifuge tube (1.7 mL)	Diamed	SPE155-N
75 cm2 tissue culture treated flask	BD biosciences	353136
Cell scraper	BD biosciences	353085
Forcepts	VWR	82027-386	fine tip, dissecting
Scissors	VWR	82027-582	Delicate, 4 1/2"
Brightfield microscope	Motic	AE31	Inverted phase contrast
Scale	Mettler	PE 3000