CD200 Mouse

What is CD200 and how is it characterized in mice?

CD200 is a 45 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. In mice, CD200 contains extracellular domains composed of one Ig-like V-type domain and one Ig-like C2-type domain, a transmembrane segment, and a short cytoplasmic domain. Within the extracellular domain, mouse CD200 shares approximately 76% amino acid sequence identity with human CD200. Western blot analysis typically detects mouse CD200 at approximately 42 kDa, particularly in mouse brain cortex tissue and various immune cells . CD200 functions primarily as an immunoregulatory molecule through interaction with its receptor CD200R, which transmits inhibitory signals to myeloid cells .

What is the expression pattern of CD200 and CD200R in mouse tissues?

CD200 demonstrates a widespread but defined distribution pattern in mice. It is prominently expressed on thymocytes, specific T cell subsets, B cells, follicular dendritic cells, endothelial cells, and neurons in brain tissue. The protein is notably absent on natural killer cells, granulocytes, monocytes, and macrophages . In contrast, CD200R expression is more restricted, with strongest expression on cells of myeloid lineage, particularly macrophages and neutrophils. Additional expression is observed on other leukocytes including monocytes, mast cells, and certain T lymphocyte subsets . This complementary distribution pattern supports CD200's role in regulating myeloid cell activity in various tissues.

How can I detect CD200 expression in mouse samples?

Multiple approaches can be employed to detect CD200 in mouse samples:

Western Blot Analysis:

• Use anti-mouse CD200 monoclonal antibodies (such as clone 325520)

• Recommended protocol: Probe PVDF membrane with 2 μg/mL of mouse anti-human/mouse CD200 antibody

• Follow with appropriate HRP-conjugated secondary antibody

• CD200 typically appears as a band at approximately 42 kDa under reducing conditions

Flow Cytometry:

• Several fluorochrome-conjugated antibodies are available, including:

  • PE anti-mouse CD200 (clone OX-90)

  • APC anti-mouse CD200 (clone OX-90)

  • Brilliant Violet 421™ anti-mouse CD200 (clone OX-90)

Immunohistochemistry:

• For frozen sections, use Alexa Fluor® 647, Alexa Fluor® 594, or purified anti-mouse CD200 antibodies

• Recommended for tissues such as spleen and brain sections

What mouse models are available for studying CD200 function?

Four primary mouse models are commonly utilized in CD200 research:

These models allow for comprehensive investigation of CD200/CD200R axis in various disease contexts and inflammatory conditions .

How should I design experiments to study CD200 function in inflammatory disease models?

When designing experiments to investigate CD200 function in inflammatory disease models, consider the following methodological approach:

• Model Selection: Choose appropriate disease models that involve myeloid cell activation, such as DSS-induced colitis, autoimmune encephalomyelitis, or arthritis models.

• Experimental Groups: Include all relevant genotypes (CD200tg, WT, CD200R1KO, CD200KO) with age-matched and sex-matched mice to ensure valid comparisons.

• Assessment Parameters:

  • Monitor clinical parameters (weight loss, disease activity scores)

  • Collect tissues for histological scoring and immunohistochemical staining

  • Isolate mononuclear cells from relevant tissues for flow cytometry

  • Prepare tissue explant cultures for cytokine measurements by ELISA

  • Perform quantitative RT-PCR for gene expression analysis

• Time Course: Include both acute and chronic phases of the disease to capture the full spectrum of CD200/CD200R involvement.

• Mechanistic Studies: Consider antibody-mediated depletion studies (e.g., anti-CD25 for Treg depletion) to further define the mechanisms underlying CD200-mediated protection .

What methods are recommended for analyzing CD200R signaling in mouse myeloid cells?

Analysis of CD200R signaling in mouse myeloid cells requires multiple complementary approaches:

• Ex vivo stimulation assays:

  • Isolate myeloid cells (macrophages, dendritic cells) from relevant mouse tissues

  • Stimulate with recombinant CD200 protein or CD200-expressing cells

  • Measure inhibition of pro-inflammatory cytokine production (TNF-α, IL-6) by ELISA

  • Compare responses between cells from WT, CD200KO, and CD200R1KO mice

• Phosphorylation studies:

  • Assess phosphorylation of downstream signaling molecules following CD200R engagement

  • Focus on known signaling pathways connected to inhibitory receptor function

• Transcriptional analysis:

  • Perform RNA-seq or qRT-PCR on myeloid cells before and after CD200 exposure

  • Identify genes differentially regulated by CD200/CD200R interaction

• Functional assays:

  • Measure phagocytic activity, oxidative burst, or migration capacity of myeloid cells

  • Compare these functions in the presence or absence of CD200 stimulation

How does the CD200/CD200R axis influence experimental colitis in mice?

The CD200/CD200R interaction plays a significant regulatory role in experimental colitis as demonstrated in DSS-induced colitis models:

• Protective effect of CD200:

  • CD200tg mice show markedly reduced sensitivity to DSS compared to WT mice

  • Protected mice exhibit lower histological scores, decreased weight loss, and reduced tissue damage

  • The protective effect is associated with attenuated infiltration of macrophages, neutrophils, and CD3+ cells

• Enhanced susceptibility in CD200/CD200R deficiency:

  • Both CD200KO and CD200R1KO mice demonstrate accelerated weight loss during acute colitis

  • These mice develop significantly higher histological scores indicative of severe colonic inflammation

  • Greater expression of macrophage-derived inflammatory cytokines is observed in colon tissue

• Mechanism of protection in chronic colitis:

  • In chronic colitis models, CD200tg mice show increased infiltration of Foxp3+ regulatory T (Treg) cells in colonic tissue

  • Protection can be attenuated by administration of anti-CD25 monoclonal antibody, suggesting Treg involvement

  • CD200 appears to inhibit inflammatory cytokine production by myeloid cells both in vivo and in vitro

This experimental evidence suggests that therapeutic approaches targeting CD200/CD200R interactions may have potential in treating inflammatory bowel diseases.

What is known about the relationship between CD200/CD200R and other immune checkpoint molecules?

The CD200/CD200R pathway represents one of several immune checkpoint mechanisms with unique features:

• Structural and functional relationships:

  • CD200R belongs to a family of genes that likely arose through gene duplication events

  • Unlike CD200R which delivers inhibitory signals, some CD200R-related molecules (CD200RLa and CD200RLb in mice) pair with DAP12, an activatory adaptor protein

  • This dual functionality resembles other immune checkpoint families such as signal regulatory proteins (SIRPs) and killer Ig-related receptors (KIRs)

• Comparative signaling mechanisms:

  • CD200R lacks traditional inhibitory motifs (like ITIMs) found in other checkpoint receptors

  • CD200R utilizes distinct signaling mechanisms that ultimately inhibit myeloid cell activation

  • This provides complementary regulation to other checkpoint pathways

• Tissue-specific functions:

  • While many checkpoint molecules function primarily within lymphoid tissues, CD200/CD200R interactions are important at tissue interfaces where myeloid regulation is critical

  • This includes the central nervous system, lungs, and intestinal mucosa

Understanding these relationships provides insight into the non-redundant functions of multiple checkpoint pathways in maintaining immune homeostasis.

How can CD200R-related genes (CD200RLa-d) be experimentally distinguished from CD200R1?

Distinguishing between CD200R1 and its related genes (CD200RLa-d) in mouse experimental systems requires specific approaches:

• Genetic analysis:

  • Use specific primers for qRT-PCR that target unique sequences in each gene

  • Sequence verification is essential due to the high homology between family members

• Protein detection:

  • Employ novel monoclonal antibodies specifically developed against each receptor variant

  • Confirm specificity using cells from corresponding knockout mice

  • When using commercially available antibodies, validate their specificity for the intended target

• Functional discrimination:

  • CD200R1 associates with inhibitory signaling pathways

  • CD200RLa and CD200RLb pair with DAP12 and deliver activating signals

  • These functional differences can be exploited in signaling assays to distinguish receptor subtypes

• Binding studies:

  • Despite substantial sequence homology with CD200R, CD200RLa and CD200RLb do not bind CD200

  • Binding assays with recombinant CD200 can help distinguish CD200R1 from related proteins

This methodological approach helps overcome the technical challenges in studying this complex receptor family.

What are common challenges in detecting mouse CD200 using antibodies and how can they be addressed?

Researchers frequently encounter specific challenges when detecting mouse CD200:

• Variable expression levels:

  • CD200 expression can be relatively low in some tissues or cell types

  • Solution: Use signal amplification techniques (e.g., biotin-streptavidin systems) for Western blotting or IHC; optimize cell isolation protocols to avoid loss of surface expression

• Cross-reactivity concerns:

  • Some antibodies may cross-react with structurally similar proteins

  • Solution: Validate antibody specificity using CD200KO mouse tissues as negative controls; for Western blots, confirm the expected molecular weight (approximately 42 kDa)

• Technical variability in flow cytometry:

  • CD200 detection can be affected by enzymatic tissue digestion methods

  • Solution: Compare results using mechanical dissociation versus enzymatic digestion; include appropriate positive controls (such as B cells or neuronal tissue)

• Fixation-sensitive epitopes:

  • Some antibody clones may perform poorly with certain fixation methods

  • Solution: Compare multiple fixation protocols (paraformaldehyde, acetone, methanol) to determine optimal conditions for your specific antibody clone

How should researchers interpret conflicting data between in vitro and in vivo CD200 studies?

When facing discrepancies between in vitro and in vivo findings in CD200 research, consider the following interpretative framework:

• Context-dependent signaling:

  • CD200/CD200R interactions occur in complex tissue microenvironments in vivo

  • In vitro systems may lack essential cellular networks and spatial organization

  • Interpretation approach: Consider in vitro findings as mechanistic insights rather than perfect predictors of in vivo outcomes

• Temporal dynamics:

  • Acute responses in vitro may differ from chronic adaptations observed in vivo

  • Interpretation approach: Design time-course experiments in vitro to better model the dynamic nature of in vivo responses

• Compensatory mechanisms:

  • Alternative pathways may compensate for CD200/CD200R deficiency in vivo but not in simplified in vitro systems

  • Interpretation approach: Analyze multiple readouts simultaneously and consider genetic redundancy

• Strain-specific differences:

  • Background strain can significantly influence CD200/CD200R biology

  • Interpretation approach: Validate key findings across multiple strains; be cautious about generalizing from a single genetic background

• Integration strategy:

  • Recommendation: Use in vivo experiments to establish physiological relevance, followed by in vitro studies to dissect specific mechanisms; return to in vivo models with targeted interventions based on in vitro findings

What considerations are important when using CD200KO or CD200R1KO mice in research?

When utilizing CD200KO or CD200R1KO mice in research, several important considerations should be addressed:

• Developmental compensation:

  • Germline knockout mice may develop compensatory mechanisms during development

  • Recommendation: Consider using inducible knockout systems or antibody blocking approaches as complementary methods

• Background strain influences:

  • The genetic background can significantly impact phenotypes in CD200/CD200R-deficient mice

  • Recommendation: Maintain knockout strains on consistent backgrounds; backcross to experimental strains for at least 10 generations; use littermate controls whenever possible

• Microbiome considerations:

  • CD200/CD200R deficiency may alter gut microbiota, which can influence experimental outcomes, particularly in inflammatory models

  • Recommendation: Co-house experimental groups; consider microbiome analysis as a potential variable, especially in colitis studies

• Age and sex considerations:

  • CD200/CD200R expression and function may vary with age and between sexes

  • Recommendation: Age-match experimental groups precisely; design studies to address potential sex differences; report both variables clearly in publications

• Validation of knockout status:

  • Incomplete deletion or unexpected expression patterns may occur

  • Recommendation: Regularly confirm knockout status using genomic PCR, RT-PCR, and protein detection methods; characterize expression in relevant tissues for your specific research question

What are promising therapeutic applications of CD200/CD200R modulation in mouse disease models?

Based on current research findings, several therapeutic applications appear promising:

• Inflammatory bowel disease:

  • CD200 overexpression provides protection in colitis models

  • Therapeutic strategy: CD200-Fc fusion proteins or agonistic anti-CD200R antibodies could mimic the protective effects of CD200tg mice

• Neuroinflammatory conditions:

  • CD200 is expressed on neurons and regulates microglial activation

  • Therapeutic strategy: Targeting CD200/CD200R axis may provide neuroprotection in models of multiple sclerosis, Parkinson's disease, or Alzheimer's disease

• Transplantation:

  • CD200/CD200R interactions help maintain immunological tolerance

  • Therapeutic strategy: Enhancing CD200 signaling could reduce graft rejection in transplantation models

• Cancer immunotherapy:

  • CD200 expression on tumor cells may suppress anti-tumor immunity

  • Therapeutic strategy: Blocking CD200/CD200R interaction could enhance immune responses against CD200+ tumors

• Autoimmune conditions:

  • The immunosuppressive effects of CD200 could be beneficial in autoimmune models

  • Therapeutic strategy: Tissue-specific delivery of CD200-based therapeutics could provide targeted immunomodulation

These applications highlight the diverse potential of CD200/CD200R modulation as a therapeutic strategy across multiple disease contexts.

How might single-cell technologies advance our understanding of CD200/CD200R biology in mice?

Single-cell technologies offer unprecedented opportunities to advance CD200/CD200R research:

• Heterogeneity in expression patterns:

  • Single-cell RNA sequencing can reveal previously unrecognized heterogeneity in CD200/CD200R expression across cell populations

  • Research opportunity: Identify novel cell types or states where CD200/CD200R signaling may be particularly important

• Temporal dynamics of signaling:

  • Single-cell signaling analysis (CyTOF, phospho-flow) can capture the kinetics of CD200R-mediated signaling at the individual cell level

  • Research opportunity: Characterize variation in signaling responses within seemingly homogeneous populations

• Spatial context:

  • Spatial transcriptomics and multiplexed imaging can map CD200/CD200R interactions within intact tissues

  • Research opportunity: Define tissue niches where CD200/CD200R interactions occur in physiological and pathological contexts

• Receptor-ligand interaction networks:

  • Single-cell multi-omics approaches can correlate CD200/CD200R expression with other immunoregulatory molecules

  • Research opportunity: Construct comprehensive interaction maps to understand how CD200/CD200R functions within broader immune regulatory networks

These technologies promise to resolve current contradictions in the field and generate new hypotheses about CD200/CD200R function in complex biological systems.