Polymorphonuclear neutrophils, or PMNs, are the vanguard of the innate immune system. They patrol the bloodstream and rush to sites of infection or injury, guided by exogenous chemotactic factors such as bacterial formyl-methionyl-leucyl-phenylalanine, known as fMLP, and by endogenous cytokines including tumor necrosis factor alpha, interleukin-1, and interleukin-8. Once they arrive, they deploy an arsenal of antimicrobial weapons: the respiratory burst, in which the enzyme NADPH oxidase generates a storm of reactive oxygen species, and the formation of neutrophil extracellular traps, web-like lattices of chromatin that ensnare bacteria. Their movements and activation states are choreographed by surface molecules such as CD11b, part of the integrin Mac-1 that drives migration into inflamed tissue; CD66b, a hallmark of neutrophil activation; and CD62L, the l-selectin that facilitates exit from the bloodstream.

In autoimmune conditions such as rheumatoid arthritis and psoriasis vulgaris, these same defense mechanisms turn against the host. For years, research into these diseases concentrated on the adaptive immune system, the domain of T and B lymphocytes. More recently, attention has shifted toward neutrophils and their capacity for reactive oxygen species generation and NETosis, both of which appear central to disease pathogenesis. Yet these conditions remain difficult to treat, and methotrexate remains one of the most important therapeutic options in rheumatoid arthritis. The drug is a competitive inhibitor of dihydrofolate reductase, blocking DNA synthesis by preventing the conversion of dihydrofolate to its active form, tetrahydrofolate. But that canonical mechanism cannot fully explain its clinical effects, since methotrexate also influences cells with low proliferative rates, including terminally differentiated neutrophils.

The research team, working at the University of Regensburg, designed their experiments around a critical methodological insight from their own prior work: the standard practice of isolating neutrophils by centrifugation markedly impairs the cells’ function, compromising migration and antigen expression, apparently because granule contents are released inside the cells during the spin. To avoid this artifact, the researchers used a gentle sedimentation approach in which whole blood was mixed with a gelatin-based sedimentation agent and left to stand, allowing red blood cells to settle while a leukocyte-rich plasma layer was collected without centrifugation of the cells themselves. Blood was drawn from healthy volunteer donors under ethical approval, yielding samples from seventeen participants, nine male and eight female, aged 22 to 59, all healthy at the time of collection.

The centerpiece of the study was a three-dimensional chemotaxis assay conducted in microfluidic-style slides from Ibidi, each containing chamber systems with two reservoirs connected by a narrow channel. The chambers were filled with a type I collagen matrix mixed with culture medium containing methotrexate at concentrations of zero, 0.5, or 5 millimoles per liter, and neutrophils suspended in fifty percent autologous plasma were introduced on one side. On the opposite side, the researchers placed either fMLP at 10 nanomolar or interleukin-8 at 100 nanograms per milliliter to establish a chemotactic gradient. The entire setup was mounted on a Leica DMi8 inverted microscope inside a climate chamber holding conditions at 37 degrees Celsius, five percent carbon dioxide, and fifty percent humidity. Over an observation period of nearly 22 hours, the camera captured one image every 30 seconds, producing roughly 2,600 images per channel.

Cell tracking was performed with Imaris software, which assigned each cell a position within a coordinate system and followed it across 60 consecutive images, corresponding to 30-minute windows. The analysis yielded several parameters, including total track length, displacement along the axis of the chemotactic gradient, and track straightness. The results were striking. With fMLP as the attractant, the median track length fell from 373.0 micrometers in untreated cells to 296.3 micrometers at 0.5 millimoles per liter methotrexate and 214.7 micrometers at 5 millimoles per liter, differences that were highly significant. But the most dramatic effect appeared in directional movement: median displacement along the gradient axis collapsed from 87.2 micrometers in controls to 45.2 and then just 11.0 micrometers at the higher concentrations. At 5 millimoles per liter, total track length retained 57.6 percent of its baseline value, yet directional displacement fell to a mere 12.6 percent.

The same pattern emerged when interleukin-8 replaced fMLP, ruling out a specific interaction between methotrexate and a single chemoattractant. Median track lengths declined from 350.7 to 326.0 and then 237.7 micrometers across the concentration series, while gradient-directed displacement plummeted from 69.5 to 50.8 and finally 10.3 micrometers, all differences statistically significant. The interpretation is subtle but important: neutrophils exposed to methotrexate continue to move, but they largely lose their compass. The drug appears to interfere with the cells’ ability to sense or respond to a chemoattractant gradient, leaving them to wander in uncoordinated, non-directional paths. Notably, the inhibitory effect on migration appeared within just a few hours, far too quickly to be explained by impaired nucleotide synthesis, since neutrophils are terminally differentiated cells with minimal de novo synthetic capacity and highly restricted transcriptional activity.

Flow cytometry experiments painted a complementary picture. The researchers incubated leukocyte-rich plasma with methotrexate at four concentrations, 0, 0.005, 0.5, and 5 millimoles per liter, chosen to span clinically relevant exposures, since low-dose regimens for rheumatoid arthritis produce micromolar plasma levels while high-dose protocols for osteosarcoma can exceed 1.5 millimoles per liter. After 60 and 240 minutes, they measured the oxidative burst using the fluorescent dye dihydrorhodamine-123 and quantified surface epitopes with fluorophore-conjugated antibodies. The activation markers CD11b, CD62L, and CD66b showed no significant changes at either time point, indicating that methotrexate neither activates nor deactivates the cells in a conventional sense. Likewise, neither reactive oxygen species production nor NETosis differed between treated and control cells, a finding consistent across both microscopy and flow cytometry, though it contrasts with an earlier study reporting reduced ROS output in neutrophils from methotrexate-treated rheumatoid arthritis patients.

The one surface molecule that did respond was LOX-1, a receptor for oxidized low-density lipoprotein known primarily from endothelial cells and macrophages but recently identified on neutrophils, where it appears to exert an immunomodulatory and potentially immunosuppressive function. LOX-1 has been proposed as a biomarker of disease activity in rheumatoid arthritis, which makes the new finding particularly consequential. After 60 minutes of incubation, LOX-1 expression at 5 millimoles per liter methotrexate reached a mean fluorescence of 145.4, significantly higher than at 0.005 and 0.5 millimoles per liter. At 240 minutes, the effect was even clearer: expression at 5 millimoles per liter was significantly elevated compared with all three lower concentrations. This means that not only rheumatoid arthritis itself but also methotrexate treatment can drive LOX-1 upregulation, raising the question of how the two factors interact in patients and whether LOX-1 measurements must be interpreted in light of therapy.

The authors are careful to note the limitations of their work. Donor-to-donor variability may have influenced the results, the in vitro assays cannot be directly extrapolated to the living body, and the experiments used neutrophils from healthy donors rather than patients with inflammatory disease, whose cells may respond differently. The chemotaxis and flow cytometry data are also largely descriptive, and the molecular mechanism behind the loss of directional sensing remains unknown. Even so, the study delivers a coherent message: methotrexate selectively disarms neutrophil chemotaxis while leaving cell viability, activation markers, ROS production, and NETosis untouched, and it concurrently raises LOX-1 expression. For a drug that has been a clinical mainstay since the 1980s yet whose full mechanism of action has never been entirely pinned down, the demonstration that it can strip immune cells of their navigational sense, independent of which chemoattractant is calling them, opens a promising avenue for understanding both the drug’s anti-inflammatory power and the biology of the cells it targets.

Subject of Research: The immunomodulatory effects of methotrexate on neutrophil chemotaxis, surface marker expression, and LOX-1 upregulation

Article Title: Methotrexate Modulates Polymorphonuclear Neutrophil Function Through Reduced Chemotaxis and Increased LOX‐1 Expression

Article References: Wieke, V., Kraus, R. F., Gruber, M. A., & Kieninger, M. (2026). Methotrexate Modulates Polymorphonuclear Neutrophil Function Through Reduced Chemotaxis and Increased LOX‐1 Expression. Immunity, Inflammation and Disease, 14(10), Article e70537. https://doi.org/10.1002/iid3.70537

Image Credits: AI Generated

DOI: 10.1002/iid3.70537

Keywords: methotrexate, neutrophils, chemotaxis, LOX-1, rheumatoid arthritis, autoimmune disease, innate immunity, flow cytometry, live-cell imaging, reactive oxygen species, NETosis, CD11b