# Controlling the Upstream Migration of Neutrophils through the Modulation of Mac-1

> **NIH NIH R21** · UNIVERSITY OF PENNSYLVANIA · 2020 · $200,965

## Abstract

Summary/Abstract
 The ability of immune cells to traffic from the blood stream to the sites of inflammation in order to find
infectious agents and transmit information to other immune cells is a hallmark of the innate immune response.
Trafficking is governed by the leukocyte adhesion cascade, a well-characterized, step-wise sequence of events
in which blood borne immune cells tether, roll, firmly arrest, migrate and then enter tissues to perform immune
cell functions. Neutrophils are the “first responders” and will traffic to the sites of inflammation immediately
upon sensing the inflammatory insult. These events normally occur within post-capillary venules, where cells
must overcome high shear rates to bind to and eventually transmigrate through the endothelial surface.
 We and others have identified an interesting phenomenon wherein certain cells of hematopoietic origin
- notably T-cells and hematopoietic stem and progenitor cells (HSPCs) – will crawl upstream, against the
direction of flow, on surfaces that contain the ligand intercellular adhesion molecule-1 (ICAM-1). This upstream
migration is mediated by the β2 integrin, αLβ2, also known as Lymphocyte Function-associated Antigen-1 (LFA-
1) binding to ICAM-1. Originally it was reported that neutrophils are unable to crawl upstream on ICAM-1, even
though neutrophils express LFA-1. Neutrophils express an additional receptor for binding ICAM-1, Mac-1,
which is up expressed when neutrophils are activated. We hypothesized that neutrophils are unable to crawl
upstream because Mac-1 dominates binding ICAM-1 in neutrophils, and that if we block or disable Mac-1, the
phenotype for upstream migration would be recovered. Our preliminary results show that by blocking Mac-1,
upstream migration of neutrophils is recovered, thus setting the premise for this application.
 In this application we will show that neutrophils can indeed crawl upstream by inhibiting interactions of
Mac-1, using both differentiated HL-60 cells and primary neutrophils. Specifically, in Aim 1, we will Determine
the conditions in which neutrophils can crawl against the direction of shear flow, using antibody
blocking to determine which integrins inhibit upstream migration in differentiated HL-60 cells and primary
human neutrophils, and confirm that blocking Mac-1 leads to upstream migration. Then, in Aim 2, we will
analyzing the altered directional migration preferences of neutrophils deficient in Mac-1, using RNA
interference to make HL-60 cell lines that are deficient in Mac-1 and LFA-1, and test their directional
preference for migration on purified molecular surfaces, as well as on stimulated HUVECs. We will validate the
results of this aim with mutants of primary neutrophils by transducing CD34+ Hematopoietic Stem cells with
shRNA against Mac-1 during their cytokine induced differentiation into neutrophils in-vitro. These studies will
ultimately identify the critical surface receptors responsible in either guiding or preventing ...

## Key facts

- **NIH application ID:** 9933030
- **Project number:** 5R21GM133060-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Daniel A Hammer
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $200,965
- **Award type:** 5
- **Project period:** 2019-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9933030

## Citation

> US National Institutes of Health, RePORTER application 9933030, Controlling the Upstream Migration of Neutrophils through the Modulation of Mac-1 (5R21GM133060-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9933030. Licensed CC0.

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