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<meta name="description" content="A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events a …"><meta name="keywords" content="pmid:33839682, PMC7948798, doi:10.1016/j.jbc.2021.100256, Research Support, Non-U.S. Gov&#x27;t, Stefan Walbaum, Benjamin Ambrosy, Peter J Hanley, Animals, Cell Membrane / metabolism*, Cells, Cultured, Humans, Immunoreceptor Tyrosine-Based Activation Motif, Macrophage-1 Antigen / metabolism*, Macrophages / metabolism*, Mice, Mice, Knockout, Phagocytosis, Pseudopodia / metabolism*, Signal Transduction, Syk Kinase / metabolism*, PubMed Abstract, NIH, NLM, NCBI, National Institutes of Health, National Center for Biotechnology Information, National Library of Medicine, MEDLINE"><meta name="robots" content="index,nofollow,noarchive"><meta property="og:title" content="Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms - PubMed"><meta property="og:url" content="https://pubmed.ncbi.nlm.nih.gov/33839682/"><meta property="og:description" content="A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events a …"><meta property="og:image" content="https://cdn.ncbi.nlm.nih.gov/pubmed/persistent/pubmed-meta-image-v2.jpg"><meta property="og:image:secure_url" content="https://cdn.ncbi.nlm.nih.gov/pubmed/persistent/pubmed-meta-image-v2.jpg"><meta property="og:type" content="website"><meta property="og:site_name" content="PubMed"><meta name="twitter:domain" content="pubmed.ncbi.nlm.nih.gov"><meta name="twitter:card" content="summary_large_image"><meta name="twitter:title" content="Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms - PubMed"><meta name="twitter:url" content="https://pubmed.ncbi.nlm.nih.gov/33839682/"><meta name="twitter:description" content="A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events a …"><meta name="twitter:image" content="https://cdn.ncbi.nlm.nih.gov/pubmed/persistent/pubmed-meta-image-v2.jpg">
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<div class="action-panel-control-wrap">
<label for="email-citation-format" class="action-panel-label">
Format:
</label>
<select id="email-citation-format" name="citation-format" class="action-panel-selector email-citation-format">
<option selected="selected" value="summary">Summary</option>
<option value="summary-text">Summary (text)</option>
<option value="abstract">Abstract</option>
<option value="abstract-text">Abstract (text)</option>
</select>
</div>
<div class="include-supplemental-container">
<input type="checkbox" aria-label="Include MeSH and other data" name="include-supplemental" id="email-include-supplemental" class="email-include-supplemental">
<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="HJy3xHB9f0TLMVQMlI43RFwWjyyd7TwDYwi8ax4vPBGfGmWCY1H5lN2ARCgp0u4X">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="HJy3xHB9f0TLMVQMlI43RFwWjyyd7TwDYwi8ax4vPBGfGmWCY1H5lN2ARCgp0u4X">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="HJy3xHB9f0TLMVQMlI43RFwWjyyd7TwDYwi8ax4vPBGfGmWCY1H5lN2ARCgp0u4X">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="HJy3xHB9f0TLMVQMlI43RFwWjyyd7TwDYwi8ax4vPBGfGmWCY1H5lN2ARCgp0u4X">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="HJy3xHB9f0TLMVQMlI43RFwWjyyd7TwDYwi8ax4vPBGfGmWCY1H5lN2ARCgp0u4X">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="33839682">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(21)00023-5"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=3048&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=33839682&amp;db=pubmed&amp;nlmid=2985121R"
title="See full text options at Elsevier Science"
data-ga-category="full_text"
data-ga-action="Elsevier Science"
data-ga-label="33839682"
><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--linkinghub.elsevier.com-ihub-images-elsevieroa.png" alt="Elsevier Science full text link"><span class="text">
Elsevier Science
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/33839682/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=33839682&amp;db=pubmed&amp;nlmid=2985121R"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="33839682"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/33839682/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/33839682/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="33839682" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F33839682%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="HJy3xHB9f0TLMVQMlI43RFwWjyyd7TwDYwi8ax4vPBGfGmWCY1H5lN2ARCgp0u4X"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
name="add-to-new-collection"
id="collections-action-dialog-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="collections_button"
data-ga-action="create_collection"
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Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms
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data-ga-label="Anne C Bachg">Anne C Bachg</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institut für Molekulare Zellbiologie, Westfälische Wilhems-Universität Münster, Münster, Germany." href="#short-view-affiliation-1" ref="linksrc=author_aff">
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data-ga-label="Markus Horsthemke">Markus Horsthemke</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institut für Molekulare Zellbiologie, Westfälische Wilhems-Universität Münster, Münster, Germany." href="#short-view-affiliation-1" ref="linksrc=author_aff">
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data-ga-label="Jeanette H W Leusen">Jeanette H W Leusen</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Center for Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands." href="#short-view-affiliation-2" ref="linksrc=author_aff">
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data-ga-label="Attila Mócsai">Attila Mócsai</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Department of Physiology, Semmelweis University School of Medicine, Budapest, Hungary." href="#short-view-affiliation-3" ref="linksrc=author_aff">
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data-ga-label="Peter J Hanley">Peter J Hanley</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institut für Molekulare Zellbiologie, Westfälische Wilhems-Universität Münster, Münster, Germany; Department of Physiology, Pathophysiology and Toxicology and ZBAF (Centre for Biomedical Education and Research), Faculty of Health, School of Medicine, Witten/Herdecke University, Witten, Germany. Electronic address: peter.hanley@uni-wh.de." href="#short-view-affiliation-4" ref="linksrc=author_aff">
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><sup class="key">1</sup> Institut für Molekulare Zellbiologie, Westfälische Wilhems-Universität Münster, Münster, Germany.</li>
<li data-affiliation-id="short-view-affiliation-2"
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><sup class="key">2</sup> Center for Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.</li>
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><sup class="key">3</sup> Department of Physiology, Semmelweis University School of Medicine, Budapest, Hungary.</li>
<li data-affiliation-id="short-view-affiliation-4"
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><sup class="key">4</sup> Institut für Molekulare Zellbiologie, Westfälische Wilhems-Universität Münster, Münster, Germany; Department of Physiology, Pathophysiology and Toxicology and ZBAF (Centre for Biomedical Education and Research), Faculty of Health, School of Medicine, Witten/Herdecke University, Witten, Germany. Electronic address: peter.hanley@uni-wh.de.</li>
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A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and KO mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80<sup>+</sup> cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcγRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.
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Keywords:
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Complement system; knockout mice; live-cell imaging; macrophages; phagocytosis.
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<p xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:p1="http://pubmed.gov/pub-one">Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.</p>
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ee79/7948798/0a896867a375/gr1.gif"
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<p> Figure 1 </p>
</strong>
<div class="figure-caption-contents"><b> Phagocytic cup formation mediated by… </b></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 1 </p>
</strong>
<div class="figure-caption-contents"><b> Phagocytic cup formation mediated by Fcγ receptors. </b> <i> A </i> , 3D time-lapse images obtained…</div>
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<figcaption id="figure-caption-0" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
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Figure 1
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<div class="figure-caption-contents"><b>Phagocytic cup formation mediated by Fcγ receptors.</b><i>A</i>, 3D time-lapse images obtained by spinning disk confocal microscopy showing a WT macrophage (Mϕ) ingesting a human red blood cell (hRBC) opsonized with mouse IgG antibodies (mouse anti-human CD235a monoclonal IgG2b antibodies). The macrophage was labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and hRBCs were labeled red fluorescent with the plasma membrane stain CellMask Orange. Scale bars, 10 μm. <i>B</i>, ingestion of an IgG-opsonized hRBC by a morphologically spherical macrophage. Note that the tip of the ingested and squeezed hRBC is pinched off during cup closure (<i>white arrow</i>). This may serve as an immune defense strategy to lyse ingested cells. Scale bar, 10 μm (grid spacing [3D view] = 3.05 μm). <i>C</i>, lack of ingestion of IgG-opsonized hRBCs by No ITAM (NOTAM) (grid spacing = 6.46 μm) and <i>Fcer1g</i><sup>/</sup> macrophages (grid spacing = 6.49 μm). NOTAM macrophages contain nonsignaling immunoreceptor tyrosine-based activation motifs (ITAMs) in the Fc receptor γ chain. <i>D</i>, cumulative plots of phagocytic (ingestion) events for WT, NOTAM, and <i>Fcer1g</i><sup>/</sup> macrophages. The plots for NOTAM and <i>Fcer1g</i><sup>/</sup> macrophages are overlaid. The <i>x</i>-axes show individual macrophages. <i>E</i>, summary box plots of phagocytic efficiency, defined as the percentage of IgG-opsonized hRBCs (in contact with a macrophage) which were fully ingested. Medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. Notably, following the introduction of IgG-opsonized hRBCs, the phagocytic efficiencies of NOTAM and <i>Fcer1g</i><sup>/</sup> macrophages were negligible, whereas WT macrophages were highly efficient eaters. Data were analyzed using the KruskalWallis one-way ANOVA (H = 89.8, degrees of freedom = 2, and <i>p</i> &lt; 0.0001). Post hoc comparisons were performed using the Dunn test; <i>n</i> (number of macrophages) = 35 (from 3 WT mice), <i>n</i> = 26 (from 4 NOTAM mice), and <i>n</i> = 35 (from 4 <i>Fcer1g</i><sup>/</sup> mice). <i>p</i> &lt; 0.0001. IgG, immunoglobulin G.</div>
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<img class="figure-thumb" itemprop="thumbnail"
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<p> Figure 2 </p>
</strong>
<div class="figure-caption-contents"><b> Ingestion of dual IgG- and… </b></div>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 2 </p>
</strong>
<div class="figure-caption-contents"><b> Ingestion of dual IgG- and complement C3b/iC3b-opsonized human red blood cells by NOTAM… </b></div>
</div>
<figcaption id="figure-caption-1" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 2
</strong>
<div class="figure-caption-contents"><b>Ingestion of dual IgG- and complement C3b/iC3b-opsonized human red blood cells by NOTAM and <i>Fcer1g</i></b><sup><b>/</b></sup><b>macrophages.</b><i>A</i>, human red blood cells (hRBCs) were dual-opsonized with IgG and complement C3b/iC3b by incubating IgG-opsonized hRBCs with complement C5-deficient (C5 null) mouse serum. <i>B</i>, macrophages isolated from No ITAM (NOTAM) mice contain nonsignaling immunoreceptor tyrosine-based activation motifs (ITAMs) in the Fc receptor γ chain, whereas the Fc receptor γ chain (encoded by <i>Fcer1g</i>) is deleted in macrophages from <i>Fcer1g</i><sup>/</sup> mice, leading to loss of expression of the α-chains FcγRI, FcγRIII, and FcγRIV. The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing FcγR, FcγRIIb, is functional in both NOTAM and <i>Fcer1g</i><sup>/</sup> macrophages. <i>C</i>, ingestion of dual IgG- and C3b/iC3b-opsonized hRBCs by NOTAM macrophages <i>via</i> phagocytic cup formation (upper panel) or partial sinking phagocytosis (lower panel). The phagocytic cups in the upper panel arose from the extension of membrane protrusions, which rolled over the particle (hRBC). Particle number 3 was not ingested because of retraction of the membrane protrusion. 3D time-lapse imaging was performed for 16 min by spinning disk confocal microscopy. Scale bars, 10 μm. <i>D</i>, ingestion of a dual IgGopsonized and C3b/iC3b-opsonized hRBC by a <i>Fcer1g</i><sup>/</sup> macrophage <i>via</i> sinking phagocytosis. Scale bar, 10 μm. <i>E</i>, cumulative plots of phagocytic (ingestion) events for NOTAM and <i>Fcer1g</i><sup>/</sup> macrophages. The <i>x</i>-axes show individual macrophages. <i>F</i>, summary box plots of macrophage phagocytic efficiency, defined as the percentage of IgG-opsonized hRBCs (in contact with a macrophage) which were fully ingested. Medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. Data were analyzed using the KruskalWallis one-way ANOVA (H = 50.4, degrees of freedom = 2, and <i>p</i> &lt; 0.0001). Post hoc comparisons were performed using the Dunn test; <i>n</i> (number of macrophages) = 35 (from 3 WT mice), <i>n</i> = 41 (from 4 NOTAM mice), and <i>n</i> = 31 (from 2 <i>Fcer1g</i><sup>/</sup> mice). <i>p</i> &lt; 0.0001. IgG, immunoglobulin G.</div>
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<strong class="figure-label">
<p> Figure 3 </p>
</strong>
<div class="figure-caption-contents"><b> Phagocytosis of dual IgM and… </b></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 3 </p>
</strong>
<div class="figure-caption-contents"><b> Phagocytosis of dual IgM and complement C3b/iC3bopsonized human red blood cells by WT… </b></div>
</div>
<figcaption id="figure-caption-2" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 3
</strong>
<div class="figure-caption-contents"><b>Phagocytosis of dual IgM and complement C3b/iC3bopsonized human red blood cells by WT and <i>Fcer1g</i></b><sup><b>/</b></sup><b>macrophages <i>via</i> sinking or phagocytic cup formation.</b><i>A</i>, ingestion of a dual IgM and complement C3b/iC3bopsonized human red blood cell (hRBC) by a WT macrophage. The macrophage was labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and the cytosol of the hRBC was loaded with the red fluorescent probe pHrodo Red. Time-lapse 3D imaging for 30 min was performed by spinning disk confocal microscopy. Particle engulfment was achieved by a tangental membrane protrusion (<i>straight white arrow</i>), which subsequently rolled over (<i>curved white arrow</i>) the hRBC. Scale bar, 10 μm. <i>B</i>, ingestion of several dual IgM and complement C3b/iC3bopsonized hRBCs by a <i>Fcer1g</i><sup>/</sup> (Fc receptor γ-chain KO) macrophage. The <i>white curved arrows</i> indicate engulfment <i>via</i> membrane protrusions rolling over the hRBC. Scale bars, 10 μm. <i>C</i>, cumulative plots of phagocytic (ingestion) events for WT and <i>Fcer1g</i><sup>/</sup> macrophages. The <i>x</i>-axes show individual macrophages. <i>D</i>, summary box plots of phagocytic efficiency, defined as the percentage of dual IgM and complement C3b/iC3bopsonized hRBCs (in contact with a macrophage), which were fully ingested. Medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. Data were analyzed using the MannWhitney U test (U = 1177.5, <i>p</i> = 0.47); <i>n</i> (number of macrophages) = 92 (from 3 WT mice) and <i>n</i> = 28 (from 2 <i>Fcer1g</i><sup>/</sup> mice). n.s., not significant (<i>p</i> &gt; 0.05). IgM, immunoglobulin M.</div>
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<img class="figure-thumb" itemprop="thumbnail"
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<p> Figure 4 </p>
</strong>
<div class="figure-caption-contents"><b> Conditional deletion of <i> Syk </i> in… </b></div>
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</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 4 </p>
</strong>
<div class="figure-caption-contents"><b> Conditional deletion of <i> Syk </i> in macrophages inhibits Fcγ receptormediated phagocytosis and phagocytic cup… </b></div>
</div>
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Figure 4
</strong>
<div class="figure-caption-contents"><b>Conditional deletion of <i>Syk</i> in macrophages inhibits Fcγ receptormediated phagocytosis and phagocytic cup formation by complement receptors.</b><i>A</i>, ingestion of a pair of IgG-opsonized human red blood cells (hRBCs) by a WT macrophage. 3D time-lapse imaging for 16 min was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and the plasma membrane was labeled red fluorescent with CellMask Orange. Scale bar, 10 μm. <i>B</i>, lack of ingestion of IgG-opsonized hRBCs by macrophages isolated from myeloid-restricted <i>Syk</i> conditional KO (cKO) mice. Scale bar, 10 μm. <i>C</i>, partial sinking of dual IgM and complement C3b/iC3bopsonized hRBCs into <i>Syk</i> cKO macrophages. The <i>white arrows</i> indicate sinking phagocytic events. Scale bars, 10 μm. <i>D</i>, summary box plots of projected cell area in WT and <i>Syk</i> cKO macrophages. The medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. Data were analyzed using the MannWhitney U test (U = 6896, <i>p</i> =0.0004); <i>n</i> (number of macrophages) = 122 (from 4 WT mice) and <i>n</i> = 88 (from 3 <i>Syk</i> cKO mice). <i>p</i> &lt; 0.001. <i>E</i>, cumulative plots of phagocytic (ingestion) events for <i>Syk</i> cKO macrophages presented with either IgG-opsonized or dual IgG and complement C3b/iC3bopsonized hRBCs. The <i>x</i>-axes show individual macrophages. IgG, immunoglobulin G.</div>
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<p> Figure 5 </p>
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<div class="figure-caption-contents"><b> Slow sinking phagocytosis mediated by… </b></div>
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<p> Figure 5 </p>
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<div class="figure-caption-contents"><b> Slow sinking phagocytosis mediated by complement receptors in <i> Syk </i> conditional KO (cKO) macrophages… </b></div>
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Figure 5
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<div class="figure-caption-contents"><b>Slow sinking phagocytosis mediated by complement receptors in <i>Syk</i> conditional KO (cKO) macrophages presented with dual IgM and complement C3b/iC3bopsonized targets.</b><i>A</i> and <i>B</i>, partial or complete ingestion of dual IgM and complement C3b/iC3bopsonized human red blood cells (hRBCs) by myeloid-restricted <i>Syk</i> conditional KO (cKO) macrophages. The <i>inset</i> in the lower panel (<i>B</i>) shows an XY optical section. 3D time-lapse imaging was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and the cytosol of hRBCs was loaded with the red fluorescent probe pHrodo Red. Scale bars, 10 μm. <i>C</i>, cumulative plots of phagocytic (ingestion) events for WT and <i>Syk</i> cKO macrophages presented with dual IgM and complement C3b/iC3bopsonized hRBCs. The <i>x</i>-axes show individual macrophages. <i>D</i>, summary box plots of phagocytic efficiency, defined as the percentage of dual IgM and complement C3b/iC3bopsonized hRBCs (in contact with a macrophage), which were fully ingested. Medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. Data were analyzed using the MannWhitney U test (U = 1002, <i>p</i> =0.6); <i>n</i> (number of macrophages) = 31 (from 3 WT mice) and <i>n</i> = 61 (from 3 <i>Syk</i> cKO mice). n.s. = not significant (<i>p</i> &gt; 0.05). IgM, immunoglobulin M.</div>
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<p> Figure 6 </p>
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<div class="figure-caption-contents"><b> Slow sinking phagocytosis in macrophages… </b></div>
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<p> Figure 6 </p>
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<div class="figure-caption-contents"><b> Slow sinking phagocytosis in macrophages lacking the ITAM adaptor Fc receptor γ chain… </b></div>
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Figure 6
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<div class="figure-caption-contents"><b>Slow sinking phagocytosis in macrophages lacking the ITAM adaptor Fc receptor γ chain and DAP12.</b><i>A</i>, slow partial sinking of dual IgG and complement C3b/iC3bopsonized human red blood cells (hRBCs) into <i>Fcer1g</i>/<i>Tyrobp</i> double KO (dKO) macrophages (Mϕs), which lack the ITAM adaptor proteins Fc receptor γ chain and DAP12. Time-lapse 3D imaging for 16 min was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and the plasma membrane was labeled red fluorescent with CellMask Orange. Scale bars, 10 μm. <i>B</i>, cumulative plots of phagocytic (ingestion) events for WT and <i>Fcer1g</i>/<i>Tyrobp</i> dKO macrophages presented with dual IgG and complement C3b/iC3bopsonized hRBCs. The <i>x</i>-axes show individual macrophages. <i>C</i>, summary box plots of phagocytic efficiency, defined as the percentage of dual IgG and complement C3b/iC3bopsonized hRBCs (in contact with a macrophage) which were fully ingested. Medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. Data were analyzed using the MannWhitney U test (U = 1465.5, <i>p</i> &lt; 0.0001); <i>n</i> (number of macrophages) = 46 (from 2 WT mice) and <i>n</i> = 35 (from 2 <i>Fcer1g</i>/<i>Tyrobp</i> dKO mice). <i>p</i> &lt; 0.0001. IgG, immunoglobulin G; ITAM, immunoreceptor tyrosine-based activation motif.</div>
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<p> Figure 7 </p>
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<div class="figure-caption-contents"><b> Ingestion of dual IgM and… </b></div>
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<p> Figure 7 </p>
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<div class="figure-caption-contents"><b> Ingestion of dual IgM and C3b/iC3bopsonized human red blood cells by <i> Fcer1g </i> /… </b></div>
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Figure 7
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<div class="figure-caption-contents"><b>Ingestion of dual IgM and C3b/iC3bopsonized human red blood cells by <i>Fcer1g</i>/<i>Tyrobp</i> double KO (dKO) macrophages.</b> Ingestion of dual IgM and complement C3b/iC3bopsonized human red blood cells (hRBCs) by <i>Fcer1g</i>/<i>Tyrobp</i> dKO macrophages (Mϕs), which lack the ITAM-containing Fc receptor γ chain and the ITAM adaptor DAP12. Time-lapse 3D imaging for 30 min was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and the cytosol of hRBCs was loaded with the red fluorescent probe pHrodo Red. Scale bars, 10 μm. IgM, immunoglobulin M; ITAM, immunoreceptor tyrosine-based activation motif.</div>
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<p> Figure 8 </p>
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<div class="figure-caption-contents"><b> Phagocytic cup formation is less… </b></div>
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<strong class="figure-label">
<p> Figure 8 </p>
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<div class="figure-caption-contents"><b> Phagocytic cup formation is less impaired in macrophages lacking <i> Tyrobp </i> (DAP12) than in… </b></div>
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Figure 8
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<div class="figure-caption-contents"><b>Phagocytic cup formation is less impaired in macrophages lacking <i>Tyrobp</i> (DAP12) than in those lacking both <i>Tyrobp</i> and <i>Fcer1g</i>.</b><i>A</i>, time-lapse 3D images obtained by spinning disk confocal microscopy showing slow sinking phagocytosis events associated with various degrees of membrane protrusive activity. <i>Tyrobp</i> (DAP12) KO (<i>Tyrobp</i><sup>/</sup>) macrophages (Mϕs) were presented with dual IgM and C3b/iC3bopsonized human red blood cells (hRBCs). Macrophages were labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and the cytosol of hRBCs was loaded with the red fluorescent probe pHrodo <i>Red</i>. The extended focus image at the top right shows resident peritoneal <i>Tyrobp</i><sup>/</sup> cells labeled with Alexa Fluor 594conjugated anti-CD11b (CD11b is a component [α-subunit] of complement receptor 3) and Alexa Fluor 488conjugated anti-F4/80 antibodies (<i>inset</i>). Nuclei (<i>blue channel</i>) were stained with Hoechst 33342, a fluorescent nucleic acid stain. Scale bars, 10 μm. <i>B</i>, cumulative plots of phagocytic (ingestion) events for WT, <i>Fcer1g</i>/<i>Tyrobp</i> double KO (dKO), and <i>Tyrobp</i><sup>/</sup> macrophages presented with dual IgM and complement C3b/iC3bopsonized hRBCs. The <i>x</i>-axes show individual macrophages. IgM, immunoglobulin M.</div>
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<p> Figure 9 </p>
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<div class="figure-caption-contents"><b> Phagocytosis of dual IgM and… </b></div>
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<p> Figure 9 </p>
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<div class="figure-caption-contents"><b> Phagocytosis of dual IgM and C3b/iC3bopsonized human red blood cells is abrogated in… </b></div>
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Figure 9
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<div class="figure-caption-contents"><b>Phagocytosis of dual IgM and C3b/iC3bopsonized human red blood cells is abrogated in <i>Itgb2</i></b><sup><b>/</b></sup><b>(complement receptor 3 [CR3] KO) macrophages.</b><i>A</i> and <i>B</i>, 3D time-lapse images obtained by spinning disk confocal microscopy showing lack of phagocytic activity in <i>Itgb2</i><sup>/</sup> (CR3 KO) macrophages, irrespective of whether cells were spread out (<i>A</i>) or rounded up (<i>B</i>). <i>Itgb2</i><sup>/</sup> (CR3 KO) macrophages (Mϕs) were presented with dual IgM and complement C3b/iC3bopsonized human red blood cells (hRBCs). Macrophages were labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and the cytosol of hRBCs was loaded with the red fluorescent probe pHrodo Red. Scale bars (<i>A</i> and <i>B</i>), 10 μm. <i>C</i>, cumulative plots of phagocytic (ingestion) events for <i>Itgb2</i><sup>/</sup> macrophages presented with either dual IgM and complement C3b/iC3bopsonized hRBCs or IgG-opsonized hRBCs. <i>D</i>, summary box plots of phagocytic efficiency, defined as the percentage of dual IgM and complement C3b/iC3bopsonized hRBCs or IgG-opsonized hRBCs (in contact with a macrophage), which were fully ingested. Medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. Data were analyzed using the KruskalWallis one-way ANOVA (H = 121.8, degrees of freedom = 3, and <i>p</i> &lt; 0.0001). Post hoc comparisons were performed using the Dunn test; <i>n</i> (number of macrophages) = 46 (from 3 WT mice), <i>n</i> = 44 (from 3 CR3 KO [<i>Itgb2</i><sup>/</sup>] mice), <i>n</i> = 52 (from 3 WT mice), and <i>n</i> = 48 (from 3 CR3 KO mice), respectively, from left to right. <i>p</i> &lt; 0.0001. IgM, immunoglobulin M; IgG, immunoglobulin G.</div>
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<p> Figure 10 </p>
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<div class="figure-caption-contents"><b> Aberrant elongated and cylindrical Fcγ</b></div>
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<p> Figure 10 </p>
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<div class="figure-caption-contents"><b> Aberrant elongated and cylindrical Fcγ receptormediated phagocytic cups in <i> Itgb2 </i> </b> <sup> <b> / </b> </sup> <b> (complement receptor… </b></div>
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Figure 10
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<div class="figure-caption-contents"><b>Aberrant elongated and cylindrical Fcγ receptormediated phagocytic cups in <i>Itgb2</i></b><sup><b>/</b></sup><b>(complement receptor 3 [CR3] KO) macrophages.</b><i>A</i>, ingestion of an IgG-opsonized human red blood cell (hRBC) by a WT macrophage. 3D time-lapse imaging was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies and the cytosol of hRBCs was loaded with the red fluorescent probe pHrodo Red. Scale bars, 10 μm. <i>B</i> and <i>C</i>, <i>Itgb2</i><sup>/</sup> (CR3 KO) macrophages ingest IgG-opsonized hRBCs <i>via</i> markedly elongated, cylindrical phagocytic cups. Scale bars, 10 μm. <i>D</i>, elongated, cylindrical phagocytic cup produced by a morphologically spherical <i>Itgb2</i><sup>/</sup> macrophage. Scale bar, 10 μm. <i>E</i>, summary box plots of projected cell area in WT and <i>Itgb2</i><sup>/</sup> macrophages. Medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. Data were analyzed using the MannWhitney U test (U = 3015, <i>p</i> =0.69); <i>n</i> (number of macrophages) = 55 (from 3 WT mice) and <i>n</i> = 114 (from 3 <i>Itgb2</i><sup>/</sup> mice). n.s., not significant (<i>p</i> &gt; 0.05). <i>F</i>, summary box plots of the maximal phagocytic cup length in WT and <i>Itgb2</i><sup>/</sup> macrophages. Data were analyzed using the MannWhitney U test (U = 290, <i>p</i> &lt; 0.0001); <i>n</i> (number of macrophages) = 62 (from 3 WT mice) and <i>n</i> = 56 (from 3 <i>Itgb2</i><sup>/</sup> mice). <i>p</i> &lt; 0.0001. IgG, immunoglobulin G.</div>
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<p> Figure 11 </p>
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<div class="figure-caption-contents"><b> Kinetics of phagocytic cup formation… </b></div>
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<p> Figure 11 </p>
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<div class="figure-caption-contents"><b> Kinetics of phagocytic cup formation and sinking phagocytosis. </b> <i> A </i> , ingestion of an…</div>
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Figure 11
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<div class="figure-caption-contents"><b>Kinetics of phagocytic cup formation and sinking phagocytosis.</b><i>A</i>, ingestion of an IgG-opsonized human red blood cell (hRBC) by a WT macrophage. 3D time-lapse imaging was performed by spinning disk confocal microscopy. Macrophages were labeled green fluorescent with Alexa Fluor 488conjugated anti-F4/80 antibodies, and the plasma membrane was labeled <i>red</i> fluorescent with CellMask Orange. Note that the contact point between the IgG-opsonized hRBC and the macrophage elicits local membrane protrusions, which mark the initiation of phagocytic cup formation. Scale bars, 10 μm. <i>B</i>, ingestion of dual IgM and complement C3b/iC3bopsonized hRBCs by a <i>Syk</i> cKO macrophage <i>via</i> slow sinking phagocytosis. Local membrane protrusions (indicated by <i>white arrows</i>) accompany sinking phagocytosis to varying extents. Scale bar, 10 μm. <i>C</i>, formation of a phagocytic cup upon a filopodium, extending from an <i>Itgb2</i><sup>/</sup> (complement receptor 3 deficient) macrophage. The <i>white arrow</i> shows thickening of the membrane at the tip of a filopodium, where it makes contact with an IgG-opsonized hRBC. The bulge of the macrophage cell body, marked by the <i>white numerical digit 1</i>, indicates where an IgG-opsonized hRBC has been fully internalized. <i>D</i>, summary box plots of ingestion times for phagocytic cup formation and sinking phagocytosis. Medians are represented by a <i>horizontal line</i> with a <i>lilac-colored circle</i>. hRBCs were singly opsonized with IgG or dual-opsonized with IgM and complement C3b/iC3b, as indicated. Data were analyzed using the KruskalWallis one-way ANOVA (H = 81.3, degrees of freedom = 3, and <i>p</i> &lt; 0.0001). Post hoc comparisons were performed using the Dunn test; <i>n</i> (number of macrophages) = 59 (from 6 WT mice), <i>n</i> = 37 (from 3 WT mice), <i>n</i> = 52 (from 3 <i>Itgb2</i><sup>/</sup> mice), and <i>n</i> = 23 (from 3 <i>Syk</i> cKO mice), respectively, from left to right. <i>p</i> &lt; 0.0001. IgM, immunoglobulin M; IgG, immunoglobulin G.</div>
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<div class="figure-caption-contents"><b> Schematic summary. </b> <i> A </i> , WT…</div>
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<div class="figure-caption-contents"><b> Schematic summary. </b> <i> A </i> , WT macrophages ingest IgG-opsonized human red blood cells (IgG-hRBCs)…</div>
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Figure 12
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<div class="figure-caption-contents"><b>Schematic summary.</b><i>A</i>, WT macrophages ingest IgG-opsonized human red blood cells (IgG-hRBCs) with high efficiency <i>via</i> rapid (23 min) phagocytic cup formation, which includes extension of ruffles, especially peripheral ruffles. Phagocytic cup formation after application of IgGRBCs is abrogated in both <i>Fcer1g</i><sup>/</sup> macrophages, which lack the Fc receptor γ chain, and NOTAM macrophages, which express a mutant Fc receptor γ chain with a nonsignaling ITAM. Phagocytic cups are also lacking in <i>Syk</i> conditional KO (cKO) macrophages. In contrast, phagocytic cups become elongated and cylindrical in <i>Itgb2</i><sup>/</sup> (complement receptor 3 [CR3] KO) macrophages presented with IgGhRBCs. A model of the signal transduction underlying Fcγ receptor (FcγR)mediated phagocytosis is shown on the <i>right</i>. Note that phagocytic cups can form upon a filopodium and cup closure can pinch off the tips of cells, as shown in the lower panel. <i>B</i>, whereas FcγRI, FcγRIII, and FcγRIV associate with the FcR γ chain, which contains an ITAM, the inhibitory FcγR (FcγRIIb) contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytosolic tail. Phagocytic cup formation was less efficient in NOTAM macrophages (which have surface expression of all four FcγRs, but lack a functional ITAM) than WT macrophages, but largely blocked in <i>Fcer1g</i><sup>/</sup> (FcR γ chain KO) macrophages, which do not express surface FcγRI, FcγRIII, and FcγRIV. Thus, in <i>Fcer1g</i><sup>/</sup> macrophages, FcγRIIb has less competition for binding to opsonic IgG, which would promote ITIM signaling, as shown in the model on the <i>right</i>. <i>C</i>, WT macrophages ingest dual IgM and complement C3b/iC3bopsonized hRBCs <i>via</i> phagocytic cup formation, extended ruffles, and varying degrees of sinking. Dual IgM and C3b/iC3bopsonized hRBCs are not ingested by <i>Itgb2</i><sup>/</sup> (CR3 KO) macrophages, implying that CR3, rather than CRIg, induces phagocytic cup formation and sinking phagocytosis. Notably, IgM, in contrast to IgG, is not recognized by Fcγ receptors. CR3-mediated phagocytic cup formation, but not sinking phagocytosis, is abrogated in macrophages lacking Syk (<i>Syk</i> conditional KO [cKO] macrophages) or the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ chain and DAP12 (<i>Fcer1g</i>/<i>Tyrobp</i> double KO [dKO] macrophages). The endocytic pathway during sinking phagocytosis is restricted in diameter and hRBCs, which are highly deformable, are squeezed into the cell interior, producing a characteristic intermediary hourglass morphology. A model of the signal transduction underlying CR3-mediated phagocytosis is shown on the <i>right</i>. IgG, immunoglobulin G; IgM, immunoglobulin M; ITAM, immunoreceptor tyrosine-based activation motif; NOTAM, No ITAM.</div>
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<div id="substances" class="substances keywords-section">
<h2 class="title">
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