How does the pigment stay under the skin?

CD64dtr mice unveil the existence of a novel CD64+ cell population in the skin that is highly granular and DT sensitive and corresponds to melanophages. (Source: https://rupress.org/jem/article/215/4/1115/42419/Unveiling-skin-macrophage-dynamics-explains-both)

The insights into how tattoo pigments stays under the skin and only dissipates and gets blurry over many years is surprisingly recent. In 2018, a team of immunologists from the Centre d'Immunologie de Marseille-Luminy in France led by Anna Baraska tested a new genetically modified mouse model that enables exploration of the human CD64 gene for immunoglobulin G receptors via exposure to diphtheria. Gene targeted mice are mice that have specific mutations to inactivate or modify a specific gene, which enables scientists to explore changes in the gene activity compared to control mice without the change. Diphtheria is a bacterial disease that affects mucous membranes of the nose and throat with a mortality rate of 5-10% of cases, particularly among young children.

Mice were tattooed on ears and tails and compared for levels of cell types. Ears had four times more melanocytes, the precursors to macrophages, than tails. They scraped the mice with diphtheria to cause tissue damage and determine how the cells appeared after healing. They found the green pigment of tattoos remained at the highest density where macrophages were concentrated and concluded that macrophages eat pigment to remove it from circulation. However, during normal cell death and replacement, cells cast off pigment that is re-engulfed by incoming cells of similar type. They witnessed this capture-release-recapture over a 90 day period and hypothesized this process is responsible for holding pigment in place, rather than removing it from the skin, as we might expect the immune system to do.

To ensure this wasn’t simply a by-product of exposure to diphtheria, they grafted the tattooed  tail skin of a gene-targeted mouse onto an albino mouse and watched the progression of healing. The tattoo remained over 6 weeks, and the cells were host cells, indicating that the host mouse’s macrophage cells were engulfing the pigment as the preceding cells died, exactly as in the original tattooed mouse.

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A follow-up study led by Helen Strandt of the University of Salzburg in Austria with members of the Marseilles group and using the same methods examined a type of structural cell called fibroblasts because they form the connective tissue of skin.  They found that fibroblasts hold pigment but occur at a lower rate than macrophage cells containing pigment. They speculate that fibroblasts are active taking up pigments during the initial administration of the tattoo when the collagen is damaged and continue to maintain pigment but at a lower rate than macrophages.

A 2022 study published in Frontiers in Immunology by a group in France specializing in the chemistry and biology of metals examined the influence of specific pigments on macrophages. Metals are at the base of several pigments, so this study involved investigation of three cobalt (purple) or cobalt alloy (blue and green) pigments and one zinc (white) pigment. The test was conducted directly on mouse macrophage cell lines and exposed to various concentrations of pigment. Pigments came from Kama Pigments and Tokyo Pigment. They compared acute exposure within the first 24 hours to a 3-day exposure recovery period. All pigments triggered macrophages to eat them, but the activity varied by pigment, and all pigments induced a slight, transitory tumor necrosis factor secretion. By contrast, blue produced a short and sustained increase in interleukin 6 secretion.

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The most recent study of the role of macrophages in holding tattoo pigment in place also examined the inflammatory and toxic roles of the inks in place. The authors, a group from Aachen, Germany led by Cheng Lin, used full-thickness 3D skin models designed to study wound healing. They found that macrophages are very efficient at taking up pigment (Sailor Jerry black), that basic black has no inflammatory or toxic effects, and that monocytes (macrophage precursors), which are particularly sensitive to absorbing pigment, and lymphocytes and granulocytes may be responsible for carrying small amounts of pigment away to the lymph nodes and internal organs.

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