In Vivo Activation of Peritoneal Macrophages in Response to Antibodies in a Murine Model

Peritoneal macrophages, residing in the peritoneal cavity engulf pathogens and secrete pro-inflammatory and anti-inflammatory cytokines to modulate the immune response.

To study in vivo activation of peritoneal macrophages, begin by restraining the mouse to minimize its movement. Take a mixture of lipopolysaccharides — pathogen-derived antigens and intravenous immunoglobulins and inject them intraperitoneally.

Post-injection, lipopolysaccharides bind to macrophage's pattern-recognition receptors, triggering an immune response and releasing pro-inflammatory cytokines, including IL-12.

In contrast, immunoglobulins interact with macrophage Fc receptors, downregulating the immune response by suppressing IL-12 production and promoting the release of anti-inflammatory cytokines, including IL-10. This co-stimulation by lipopolysaccharides and immunoglobulins activates peritoneal macrophages into anti-inflammatory macrophages.

Secure the euthanized mouse and make a longitudinal incision along the abdominal cavity. Retract the skin from the abdominal muscle. Inject a buffer into the peritoneal cavity; agitate to dislodge cells from the peritoneal cavity.

Collect the activated macrophage-containing peritoneal fluid.

Culture these activated macrophages in a medium containing co-stimulatory molecules — immunoglobulins, and lipopolysaccharides to enable cytokine production. Collect the spent medium and quantify cytokine levels using an enzyme-linked immunosorbent assay.

A higher IL-10 concentration than IL-12 confirms successful peritoneal anti-inflammatory macrophage activation.

Prepare a 1-milliliter syringe with the required amount of intravenous immunoglobulin and LPS, and attach a 26-gauge needle. For controls, use PBS plus LPS instead.

Take hold of the mouse by its scruff while securing its hindquarters and tail using one hand. Target the lower-right quadrant of the abdomen, and insert the needle at a 30 to 40-degree angle with the bevel up, and insert it about 1.5 cm. Then, inject the bolus. After an hour, harvest peritoneal macrophages after euthanizing the mouse.

In a sterile hood, pin the euthanized mouse to a foam board with limbs spread out, and sterilize the abdomen with 70% ethanol. Then, make a shallow 2-millimeter incision along the midline, and pull off the abdominal skin. Avoid puncturing the peritoneal wall.

Next, fully load a 5-milliliter syringe with sterile PBS at pH 7.4, and inject the mouse using a 25 or 27-gauge needle at the top of the peritoneal cavity from either side toward the midline with the bevel of the needle up. After pulling the needle out, massage the peritoneum for 10 seconds to dislodge the cells.

Now, insert the needle again back into the cavity, and collect about 3.75 milliliters of lavage fluid. Transfer the lavage fluid to a 15-milliliter conical tube on ice. Repeat the injection, massage, and collection of lavage fluid three more times, and pool all the collections into the same tube.

During the final collection, it may be easier to withdraw the lavage fluid from the far side of the mouse. Next, spin down the cells, and resuspend them in 500 microliters of plating medium. Then, count the viable macrophages, and resuspend them at 1 million per milliliter. Now, plate 100 microliters of suspension into the wells of a 96-well flat-bottom plate. Next, incubate the cells for an hour.

The peritoneal macrophages will adhere to the plate. Then, remove the non-adherent cells, and rinse each well twice with 200 microliters of warm IMDM. After each wash, wait 10 seconds, and slowly tilt the plate to pool the rinse solution. After the washes, add back 100 microliters of plating medium and place the plate in the incubator.

For the ELISA, continue to incubate the cells without stimulation for 24 hours. Then, collect and clarify the conditioned medium for cytokine analysis.