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1.  CD23+/CD21hi B-cell translocation and ipsilateral lymph node collapse is associated with asymmetric arthritic flare in TNF-Tg mice 
Arthritis Research & Therapy  2011;13(4):R138.
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints. However, how arthritic flare occurs only in select joints during a systemic autoimmune disease remains an enigma. To better understand these observations, we developed longitudinal imaging outcomes of synovitis and lymphatic flow in mouse models of RA, and identified that asymmetric knee flare is associated with ipsilateral popliteal lymph node (PLN) collapse and the translocation of CD23+/CD21hi B-cells (B-in) into the paracortical sinus space of the node. In order to understand the relationship between this B-in translocation and lymph drainage from flaring joints, we tested the hypothesis that asymmetric tumor necrosis factor (TNF)-induced knee arthritis is associated with ipsilateral PLN and iliac lymph node (ILN) collapse, B-in translocation, and decreased afferent lymphatic flow.
TNF transgenic (Tg) mice with asymmetric knee arthritis were identified by contrast-enhanced (CE) magnetic resonance imaging (MRI), and PLN were phenotyped as "expanding" or "collapsed" using LNcap threshold = 30 (Arbitrary Unit (AU)). Inflammatory-erosive arthritis was confirmed by histology. Afferent lymphatic flow to PLN and ILN was quantified by near infrared imaging of injected indocyanine green (NIR-ICG). The B-in population in PLN and ILN was assessed by immunohistochemistry (IHC) and flow cytometry. Linear regression analyses of ipsilateral knee synovial volume and afferent lymphatic flow to PLN and ILN were performed.
Afferent lymph flow to collapsed nodes was significantly lower (P < 0.05) than flow to expanding nodes by NIR-ICG imaging, and this occurred ipsilaterally. While both collapsed and expanding PLN and ILN had a significant increase (P < 0.05) of B-in compared to wild type (WT) and pre-arthritic TNF-Tg nodes, B-in of expanding lymph nodes (LN) resided in follicular areas while B-in of collapsed LN were present within LYVE-1+ lymphatic vessels. A significant correlation (P < 0.002) was noted in afferent lymphatic flow between ipsilateral PLN and ILN during knee synovitis.
Asymmetric knee arthritis in TNF-Tg mice occurs simultaneously with ipsilateral PLN and ILN collapse. This is likely due to translocation of the expanded B-in population to the lumen of the lymphatic vessels, resulting in a dramatic decrease in afferent lymphatic flow. PLN collapse phenotype can serve as a new biomarker of knee flare.
PMCID: PMC3239381  PMID: 21884592
2.  Power Doppler Ultrasound Phenotyping of Expanding versus Collapsed Popliteal Lymph Nodes in Murine Inflammatory Arthritis 
PLoS ONE  2013;8(9):e73766.
Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic “expanding” phase of the disease, and then suddenly “collapse” during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553±0.007 vs. 0.008±0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.
PMCID: PMC3767819  PMID: 24040061
3.  Efficacy of B cell Depletion Therapy on Joint Flare is Associated with Increased Lymphatic Flow 
Arthritis and rheumatism  2013;65(1):130-138.
B cell depletion therapy (BCDT) ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritic flare in tumor necrosis factor transgenic (TNF-Tg) mice is associated with efferent lymph node (LN) “collapse,” triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. We examined whether BCDT efficacy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells.
We developed contrast-enhancement (CE) MRI imaging, near-infrared indocyanine green (NIR-ICG) imaging, and intravital immunofluorescent imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-Tg mice. We tested to see if BCDT efficacy is associated with restoration of lymphatic draining and cell egress from arthritic joints.
Unlike active lymphatics to normal and pre-arthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescent imaging demonstrated that CD11b+ monocytes/macrophages in lymphatic vessels afferent to expanding LN travel at high velocity (186 ± 37 micrometer/sec), while these cells are stationary in lymphatic vessels afferent to collapsed PLN. BCDT of flaring TNF-Tg mice significantly decreased knee synovial volume by 50% from the baseline level, and significantly increased lymphatic clearance versus placebo (p<0.05). This increased lymphatic drainage restored macrophages egress from inflamed joints without recovery of the lymphatic pulse.
These results support a novel mechanism in which BCDT of flaring joints lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.
PMCID: PMC3535508  PMID: 23002006
Rheumatoid Arthritis (RA); Flare; Tumor Necrosis Factor (TNF); B cells in Inflamed Lymph Nodes (B-in); Lymphatic Pulse
4.  Near infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute vs. chronic phases of arthritis in mice 
Arthritis and rheumatism  2010;62(7):1881-1889.
Development of an in vivo imaging method to assess lymphatic draining function in the K/B×N mouse model of inflammatory arthritis.
Indocyanine green (ICG), a near-infrared (NIR) fluorescent dye, was injected intradermally into the footpad of wild-type mice, the limb was illuminated with an 806 nm NIR laser, and the movement of ICG from the injection site to the draining popliteal lymph node (PLN) was recorded with a CCD camera. ICG-NIR images were analyzed to obtain 5 measures of lymphatic function across time. K/B×N arthritic mice and control non-arthritic littermates were imaged at one-month of age when acute joint inflammation commenced, and repeated at 3 months when joint inflammation became chronic. Lymphangiogenesis in PLNs was assessed by immunochemistry.
ICG and its transport within lymphatic vessels were readily visualized and quantitative measures derived. During the acute phase of arthritis, the lymphatic vessels were dilated with increased ICG signal intensity and lymphatic pulses, and PLNs became fluorescent quickly. During the chronic phase, new lymphatic vessels were present near the foot. However, ICG appearance in lymphatic vessels was delayed. The size and area of PLN lymphatic sinuses progressively increased in the K/B×N mice.
ICG-NIR lymphatic imaging is a valuable method to assess the lymphatic draining function in mice with inflammatory arthritis. ICG-NIR imaging of K/B×N mice identified two distinct lymphatic phenotypes during the acute and chronic phase of inflammation. This technique can be used to assess new therapies for lymphatic disorders.
PMCID: PMC2902657  PMID: 20309866
Near infrared; lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
5.  Expanded CD23+/CD21hi B cells in inflamed lymph nodes are associated with the onset of inflammatory-erosive arthritis in TNF-transgenic mice and are targets of anti-CD20 therapy1 
Anti-CD20 B cell depletion therapy (BCDT) is very effective for some patients with rheumatoid arthritis (RA), however the pathogenic role of B lymphocytes in RA and the primary targets of BCDT are unknown. The human TNF transgenic (hTNF-tg) mouse model of RA displays a chronic-progressive disease that spreads from distal to proximal joints, and is generally considered to be adaptive immune system-independent. We have previously reported that knee arthritis in hTNF-tg mice is accompanied by structural and functional changes of the adjoining popliteal lymph node (PLN), detectable by contrast-enhanced magnetic resonance imaging (CE-MRI). To better understand these changes, here we show that onset of knee synovitis and focal erosions are paralleled by PLN contraction and accumulation of large numbers of B cells in the lymphatic sinus spaces within the node. Flow cytometry from 2, 4-5, and 8-12 month old TNF-tg mice demonstrated that B cell accumulation in the PLN follows ankle arthritis, but commences before knee disease, and involves early expansion of CD21hi, CD23+, IgMhi, CD1d+, activation marker-negative, polyclonal B cells which are found to be specifically restricted to lymph nodes draining inflamed, arthritic joints. The same B cell population also accumulates in PLNs of K/BxN mice with autoantigen-dependent arthritis. Strikingly, we show that BCDT ameliorates hTNF-tg disease and clears follicular and CD21hi, CD23+ B cells from the PLNs. Based on these findings, we propose a model whereby B cells contribute to arthritis in mice, and possibly RA, by directly affecting the structure, composition and function of joint-draining lymph nodes.
PMCID: PMC2874087  PMID: 20435928
B-cells; Inflammation; Rheumatoid Arthritis; Lymph nodes; B cell depletion therapy
6.  Inhibition of lymphangiogenesis and lymphatic drainage via VEGFR-3 blockade increases the severity of inflammation in chronic inflammatory arthritis 
Arthritis and rheumatism  2009;60(9):2666-2676.
Investigation of the effect of lymphatic inhibition on joint and draining lymph node pathology during the course of arthritis progression in mice.
TNF transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Mice received contrast enhanced MRI to obtain ankle and knee joint synovial volumes and draining popliteal lymph node (PLN) volumes before and 8 weeks after treatment with VEGFR-3 or VEGFR-2 neutralizing antibodies, or isotype IgG. The animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining PLNs prior to sacrifice. Lymphatic vessel formation and morphology of joints and PLNs were examined by histology, immunohistochemistry, and RT-PCR.
Compared to IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of PLNs, the number of lymphatic vessels in joints and PLNs, the lymphatic drainage from paws to PLNs, and the number of VEGF-C expressing CD11b+ myeloid cells in PLNs. However, it increased the synovial volumes and inflammatory area in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation.
Lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.
PMCID: PMC2810533  PMID: 19714652
Lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
7.  Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages. 
Infection and Immunity  1993;61(8):3244-3249.
We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.
PMCID: PMC280995  PMID: 8335357
8.  Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging 
Biomedical Optics Express  2013;4(6):967-977.
The lymphatic system provides an initial route for cancer cell dissemination in many cancers including melanoma. However, it is largely unknown how the lymphatic system changes during tumor progression due in part to the lack of imaging techniques currently available. In this study, we non-invasively imaged changes of lymphatic function and drainage patterns using near-infrared fluorescence (NIRF) imaging. Dynamic NIRF imaging following intradermal injection of indocyanine green (ICG) was conducted in C57BL/6 mice prior to inoculation of B16F10 murine melanoma cells to the dorsal aspect of the left hindpaw for baseline data or directly to the popliteal lymph node (PLN) and until 21 days post-implantation (p.i.). A series of acquired fluorescent images were quantified to measure lymphatic contractile function. Computed tomography (CT) was also performed to measure the volume of tumor-draining lymph nodes (LNs). We observed significant reduction of lymphatic contractility from 7 days p.i. until 21 days p.i.. Altered lymphatic drainage patterns were also detected at 21 days p.i. in mice with tumor in the paw and at 11 days p.i. in mice with tumor in the PLN, due to lymphatic obstruction of normal lymphatic drainages caused by extensive tumor invasion of draining LNs. Since lymphatic function and architecture were progressively altered during tumor growth and metastasis, non-invasive NIRF imaging may provide a new method to stage disease. In addition, this novel technique can be used as a diagnostic method to non-invasively assess lymphatic response as mechanism of therapeutic action.
PMCID: PMC3675875  PMID: 23761026
(170.0170) Medical optics and biotechnology; (170.2655) Functional monitoring and imaging; (170.3880) Medical and biological imaging; (170.4580) Optical diagnostics for medicine
9.  Mouse Model of Lymph Node Metastasis via Afferent Lymphatic Vessels for Development of Imaging Modalities 
PLoS ONE  2013;8(2):e55797.
Animal studies of lymph node metastasis are constrained by limitations in the techniques available for noninvasive monitoring of the progression of lymph node metastasis, as well as difficulties in the establishment of appropriate animal models. To overcome these challenges, this study has developed a mouse model of inter-lymph-node metastasis via afferent lymphatic vessels for use in the development of imaging modalities. We used 14- to 18-week-old MRL/MpJ−/lpr/lpr (MRL/lpr) mice exhibiting remarkable systemic lymphadenopathy, with proper axillary lymph nodes (proper-ALNs) and subiliac lymph nodes (SiLNs) that are 6 to 12 mm in diameter (similar in size to human lymph nodes). When KM-Luc/GFP malignant fibrous histiocytoma-like cells stably expressing the firefly luciferase gene were injected into the SiLN, metastasis could be detected in the proper-ALN within 3 to 9 days, using in vivo bioluminescence imaging. The metastasis route was found to be via the efferent lymphatic vessels of the SiLN, and metastasis incidence depended on the number of cells injected, the injection duration and the SiLN volume. Three-dimensional contrast-enhanced high-frequency ultrasound imaging showed that the blood vessel volume and density in the metastasized proper-ALN significantly increased at 14 days after tumor cell inoculation into the SiLN. The present metastasis model, with lymph nodes similar in size to those of humans, has potential use in the development of ultrasound imaging with high-precision and high-sensitivity as well as other imaging modalities for the detection of blood vessels in lymph nodes during the progression of metastasis.
PMCID: PMC3565997  PMID: 23405215
10.  Impact of Molecular Weight on Lymphatic Drainage of a Biopolymer-Based Imaging Agent 
Pharmaceutics  2012;4(2):276-295.
New lymphatic imaging technologies are needed to better assess immune function and cancer progression and treatment. Lymphatic uptake depends mainly on particle size (10–100 nm) and charge. The size of carriers for imaging and drug delivery can be optimized to maximize lymphatic uptake, localize chemotherapy to lymphatic metastases, and enable visualization of treatment deposition. Toward this end, female BALB/c mice were injected subcutaneously in the hind footpad or forearm with a series of six different molecular weight hyaluronan (HA) near-infrared dye (HA-IR820) conjugates (ca. 5–200 nm). Mice were imaged using whole body fluorescent imaging over two weeks. HA-IR820 fluorescence was clearly visualized in the draining lymphatic capillaries, and in the popliteal and iliac or axillary lymph nodes. The 74-kDa HA-IR820 had the largest lymph node area-under-the-curve. In contrast to prior reports, mice bearing limb tumors exhibited three-fold longer retention of 74-kDa HA-IR820 in the popliteal node compared to mice without tumors. HA conjugate kinetics and disposition can be specifically tailored by altering their molecular weight. The specific lymphatic uptake and increased nodal retention of HA conjugates indicate significant potential for development as a natural biopolymer for intralymphatic drug delivery and imaging.
PMCID: PMC3834911  PMID: 24300232
lymphatic imaging; hyaluronan; fluorescence; nanoparticle; tumor metastasis
11.  Migration patterns of dendritic cells in the mouse. Traffic from the blood, and T cell-dependent and -independent entry to lymphoid tissues 
Dendritic cells (DC) are critical accessory cells for primary immune responses and they may be important stimulators of transplantation reactions, but little is known of their traffic into the tissues. We have studied the migration of purified splenic DC and T lymphocytes, labeled with 111Indium-tropolone, in syngeneic and allogeneic mice. First we demonstrate that DC can migrate from the blood into some lymphoid and nonlymphoid tissues. Immediately after intravenous administration, radio-labeled DC were sequestered in the lungs, but they actively migrated into the liver and spleen and reached equilibrium levels between 3 and 24 h after transfer. At least half of the radiolabel accumulated in the liver, but the spleen was the principal site of DC localization in terms of specific activity (radiolabel per weight of tissue). DC were unable to enter Peyer's patches, or mesenteric and other peripheral lymph nodes from the bloodstream. This was also true in splenectomized recipients, where the otherwise spleen-seeking DC were quantitatively diverted to the liver. In contrast, T cells homed readily to the spleen and lymph nodes of normal mice and increased numbers were present in these tissues in splenectomized mice. Thus, unlike T cells, DC cannot recirculate from blood to lymph via the nodes. We then show that migration of DC from the blood into the spleen is dependent on the presence of T cells: DC did not enter the spleens of nude mice, but when they were reconstituted with T cells the numbers entering the spleen resembled those in euthymic mice. In nude mice, as in splenectomized recipients, the DC that would normally enter the spleen were quantitatively diverted to the liver. These findings suggest that there is a spleen- liver equilibrium for DC, that may be akin to that existing between spleen and lymph node for T cells. Finally, we followed the traffic of radiolabeled DC via the afferent lymphatics after subcutaneous footpad inoculation. DC accumulated in the popliteal nodes but did not migrate further to the inguinal nodes. There was no difference between euthymic and nude mice, showing that unlike traffic to the spleen, this route probably does not require T cells. These migration patterns were not affected by major histocompatibility barriers, and were only seen with viable, but not glutaraldehyde-fixed, DC.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2188851  PMID: 3258009
Physics in medicine and biology  2013;58(5):10.1088/0031-9155/58/5/N59.
We developed models of lymphatic nodes for 6 pediatric and 2 adult hybrid computational phantoms to calculate the lymphatic node dose estimates from external and internal radiation exposures. We derived the number of lymphatic nodes from the recommendations in International Commission on Radiological Protection (ICRP) Publications 23 and 89 at 16 cluster locations for the lymphatic nodes: extrathoracic, cervical, thoracic (upper and lower), breast (left and right), mesentery (left and right), axillary (left and right), cubital (left and right), inguinal (left and right), and popliteal (left and right), for different ages (newborn, 1-, 5-, 10-, 15-year-old, and adult). We modeled each lymphatic node within the voxel format of the hybrid phantoms by assuming that all nodes have identical size derived from published data except narrow cluster sites. The lymph nodes were generated by the following algorithm: (1) selection of the lymph node site among the 16 cluster sites; (2) random sampling of the location of the lymph node within a spherical space centered at the chosen cluster site; (3) creation of the sphere or ovoid of tissue representing the node based on lymphatic node characteristics defined in ICRP Publications 23 and 89. We created lymph nodes until the pre-defined number of lymphatic nodes at the selected cluster site was reached. This algorithm was applied to pediatric (newborn, 1-, 5-, and 10-year-old male, and 15-year-old males) and adult male and female ICRP-compliant hybrid phantoms after voxelization. To assess the performance of our models for internal dosimetry, we calculated dose conversion coefficients, called S values, for selected organs and tissues with Iodine-131 distributed in 6 lymphatic node cluster sites using MCNPX2.6, a well validated Monte Carlo radiation transport code. Our analysis of the calculations indicates that the S values were significantly affected by the location of the lymph node clusters and that the values increased for smaller phantoms due to the shorter inter-organ distances compared to the bigger phantoms. By testing sensitivity of S values to random sampling and voxel resolution, we confirmed that the lymph node model is reasonably stable and consistent for different random samplings and voxel resolutions.
PMCID: PMC3878984  PMID: 23391692
lymphatic node; computational hybrid phantom; Monte Carlo transport; S value
13.  Spatio-Temporal Changes of Lymphatic Contractility and Drainage Patterns following Lymphadenectomy in Mice 
PLoS ONE  2014;9(8):e106034.
To investigate the redirection of lymphatic drainage post-lymphadenectomy using non-invasive near-infrared fluorescence (NIRF) imaging, and to subsequently assess impact on metastasis.
Cancer-acquired lymphedema arises from dysfunctional fluid transport after lymphadenectomy performed for staging and to disrupt drainage pathways for regional control of disease. However, little is known about the normal regenerative processes of the lymphatics in response to lymphadenectomy and how these responses can be accelerated, delayed, or can impact metastasis.
Changes in lymphatic “pumping” function and drainage patterns were non-invasively and longitudinally imaged using NIRF lymphatic imaging after popliteal lymphadenectomy in mice. In a cohort of mice, B16F10 melanoma was inoculated on the dorsal aspect of the paw 27 days after lymphadenectomy to assess how drainage patterns affect metastasis.
NIRF imaging demonstrates that, although lymphatic function and drainage patterns change significantly in early response to popliteal lymph node (PLN) removal in mice, these changes are transient and regress dramatically due to a high regenerative capacity of the lymphatics and co-opting of collateral lymphatic pathways around the site of obstruction. Metastases followed the pattern of collateral pathways and could be detected proximal to the site of lymphadenectomy.
Both lymphatic vessel regeneration and co-opting of contralateral vessels occur following lymphadenectomy, with contractile function restored within 13 days, providing a basis for preclinical and clinical investigations to hasten lymphatic repair and restore contractile lymphatic function after surgery to prevent cancer-acquired lymphedema. Patterns of cancer metastasis after lymphadenectomy were altered, consistent with patterns of re-directed lymphatic drainage.
PMCID: PMC4149501  PMID: 25170770
14.  Lymphangiogenesis in Regional Lymph Nodes Is an Independent Prognostic Marker in Rectal Cancer Patients after Neoadjuvant Treatment 
PLoS ONE  2011;6(11):e27402.
One of the major prognostic factors in rectal cancer is lymph node metastasis. The formation of lymph node metastases is dependent on the existence of a premetastatic niche. An important factor preceding metastasis are lymph vessels which are located in the lymph node. Accordingly, the occurrence of intranodal lymphangiogenesis is thought to indicate distant metastasis and worse prognosis. To evaluate the significance of lymph node lymphangiogenesis, we studied formalin fixed, paraffin embedded adenocarcinomas and regional lymph nodes of 203 rectal cancer patients who were treated with neoadjuvant radiochemotherapy and consecutive curative surgery with cancer free surgical margins (R0). Regional lymph node lymph vessels were detected by immunohistochemistry for podoplanin (D2-40). Our results show that the presence of lymphatic vessels in regional lymph nodes significantly affects the disease-free survival in univariate and multivariate analyses. In contrast, there was no correlation between peritumoral or intratumoral lymph vessel density and prognosis. Indeed, our study demonstrates the importance of lymphangiogenesis in regional lymph nodes after neoadjuvant radiochemotherapy and consecutive surgery as an independent prognostic marker. Staining for intranodal lymphangiogenesis and methods of intravital imaging of lymphangiogenesis and lymphatic flow may be a useful strategy to predict long-term outcome in rectal cancer patients. Furthermore, addition of VEGF-blocking agents to standardized neoadjuvant treatment schemes might be indicated in advanced rectal cancer.
PMCID: PMC3210168  PMID: 22087309
15.  Convective diffusion of nanoparticles from the epithelial barrier towards regional lymph nodes 
Drug delivery using nanoparticles as drug carriers has recently attracted the attention of many investigators. Targeted delivery of nanoparticles to lymph nodes is especially important to prevent cancer metastasis or infection, and to diagnose disease stage. However, systemic injection of nanoparticles often results in organ toxicity because they reach and accumulate in all the lymph nodes in the body. An attractive strategy would be to deliver the drug-loaded nanoparticles to a subset of draining lymph nodes corresponding to a specific site or organ to minimize systemic toxicity. In this respect, mucosal delivery of nanoparticles to regional draining lymph nodes of a selected site creates a new opportunity to accomplish this task with minimal toxicity. One example is the delivery of nanoparticles from the vaginal lumen to draining lymph nodes to prevent the transmission of HIV in women. Other known examples include mucosal delivery of vaccines to induce immunity. In all cases, molecular and particle transport by means of diffusion and convective diffusion play a major role. The corresponding transport processes have common inherent regularities and are addressed in this review. Here we use nanoparticles delivery from the vaginal lumen to lymph nodes as an example to address the many aspects of associated transport processes. In this case, nanoparticles penetrate the epithelial barrier and move through the interstitium (tissue) to the initial lymphatics until they finally reach the lymph nodes.
Since the movement of interstitial liquid near the epithelial barrier is retarded, nanoparticles transport was found to take place through special foci present in the epithelium. Immediately after nanoparticles emerge from the foci, they move through the interstitium due to diffusion affected by convection (convective diffusion). Specifically, the convective transport of nanoparticles occurs due to their convection together with interstitial fluid through the interstitium towards the initial lymph capillaries. Afterwards, nanoparticles move together with the lymph flow along the initial lymph capillaries and then enter the afferent lymphatics and ultimately reach the lymph node. As the liquid moves through the interstitium towards the initial lymph capillaries due to the axial movement of lymph along the lymphatics, the theory for coupling between lymph flow and concomitant flow through the interstitium is developed to describe this general case.
The developed theory is applied to interpret the large uptake of Qdots by lymph nodes during inflammation, which is induced by pre-treating mouse vagina with the surfactant Nonoxynol-9 prior to instilling the Qdots. Inflammation is viewed here to cause broadening of the pores within the interstitium with the concomitant formation of transport channels which function as conduits to transport the nanoparticles to the initial lymph capillaries. We introduced the term “effective channels” to denote those channels which interconnect with foci present in the epithelial barrier and which function to transport nanoparticles to initial lymph capillaries. The time of transport towards the lymph node, predicated by the theory, increases rapidly with increasing the distance y0 between the epithelial barrier and the initial lymph capillaries. Transport time is only a few hours, when y0 is small, about some R (where R is the initial lymph capillary radius), due to the predomination of a rather rapid convection in this case. This transport time to lymph nodes may be tens of hours (or longer) when y0 is essentially larger and the slow diffusion controls the transport rate in a zone not far from the epithelial barrier, where convection is weak at large y0. Accounting for transport by diffusion only, which is mainly considered in many relevant publications, is not sufficient to explain our nanoparticles uptake kinetics because the possibility of fast transport due to convection is overlooked. Our systematic investigations have revealed that the information about the main transport conditions, namely, y0 and the pore broadening up to the dimension of the interstitial transport channels, is necessary to create the quantitative model of enhanced transport during inflammation with the use of the proposed model as a prerequisite.
The modeling for convective diffusion of nanoparticles from the epithelial barrier to the lymph node has been mainly accomplished here, while the diffusion only scenario is accounted for in other studies. This first modeling is a semi-quantitative one. A more rigorous mathematical approach is almost impossible at this stage because the transport properties of the model are introduced here for the first time. These properties include: discovery of foci in the epithelium, formation of transport channels, definition of channels interconnecting with foci (effective foci and channels), generation of flow in the interstitium towards the initial lymph capillaries due to axial flow within afferent lymphatics, deformation of this flow due to hydrodynamic impermeability of the squamous layer with the formation of the hydrodynamic stagnation zone near the epithelial barrier, predomination of slow diffusion transport within the above zone, and predomination of fast convection of nanoparticles near the initial lymph capillaries.
PMCID: PMC3804055  PMID: 23859221
16.  Lymph-node staining with activated carbon CH40: a new method for axillary lymph-node dissection in breast cancer 
Canadian Journal of Surgery  2000;43(3):191-196.
To demonstrate the usefulness of activated carbon particles (CH40) as a vital staining dye for visualizing lymphatic vessels and lymph nodes in breast cancer.
A retrospective evaluation.
Department of Surgery in Sendai National Hospital, Japan, a 716-bed teaching hospital.
To identify as many lymph nodes as possible in the axillary fat, by which we might decrease the possibility of the presence of undetected metastatic nodes, an emulsion of activated carbon particles (CH40) was injected into the centre of the mammary gland, close to the tumour site, 3 days before radical surgery.
Main outcome measure
The number of lymph nodes found by the traditional method and by the CH40-injection method were recorded.
After injection, the CH40 was readily adsorbed into regional lymphatics and streamed along with the lymph flow to blacken regional lymph nodes. The CH40-guided method increased the mean number of nodes per case found in the axilla from 8.4, by the traditional method, to 14.0 nodes per case.
The use of the CH40 technique has two technical advantages; one is that it allows surgeons to locate the blackened lymph nodes at the time of surgery and the other is that it allows pathologists to look for the nodes in fatty tissue. Lymph-node dissection with the aid of activated carbon particles is inexpensive, easy to perform and enables the smallest lymph nodes to be easily recognized. CH40 is the technique of choice for the detection of axillary lymph nodes in cases where the number of lymph nodes detected by the traditional method is too small for accurate surgery. In conclusion, the present study demonstrates that CH40 could be an appropriate tool for more accurate staging of breast cancer axillary specimens.
PMCID: PMC3695160  PMID: 10851412
17.  Response in distant lymph nodes of mice to infection in the hind footpad with Mycobacterium marinum. 
Infection and Immunity  1980;28(1):225-229.
In an attempt to demonstrate the importance of the popliteal lymph node in limiting the progress of infection with Mycobacterium marinum in the hind footpads of C57BL mice, such infections were studied in mice subjected to popliteal or popliteal and inguinal adenectomies. In the absence of the popliteal node, the footpad infection was only slightly enhanced compared with infections of sham-operated control mice; the inguinal node was found to be greatly enlarged and appeared to have substituted for the absent popliteal node. In the absence of both popliteal and inguinal nodes, the disease process in the footpads was again only slightly enhanced, and the axillary node appeared to have enlarged greatly and to have functionally replaced the missing, more proximate nodes. In additional experiments, mice subjected to adenectomy only on one side and injected in that hind footpad with phytohemagglutinin or India ink demonstrated hypertrophy or deposition of carbon particles in the more distant node only on the side of the injection. Thus, there appear to be rather direct functional connections among popliteal, inguinal, and axillary nodes that do not depend on blood circulation.
PMCID: PMC550916  PMID: 7380564
18.  Quantum dots in axillary lymph node mapping: Biodistribution study in healthy mice 
BMC Cancer  2008;8:111.
Breast cancer is the first cause of cancer death among women and its incidence doubled in the last two decades. Several approaches for the treatment of these cancers have been developed. The axillary lymph node dissection (ALND) leads to numerous morbidity complications and is now advantageously replaced by the dissection and the biopsy of the sentinel lymph node. Although this approach has strong advantages, it has its own limitations which are manipulation of radioactive products and possible anaphylactic reactions to the dye. As recently proposed, these limitations could in principle be by-passed if semiconductor nanoparticles (quantum dots or QDs) were used as fluorescent contrast agents for the in vivo imaging of SLN. QDs are fluorescent nanoparticles with unique optical properties like strong resistance to photobleaching, size dependent emission wavelength, large molar extinction coefficient, and good quantum yield.
CdSe/ZnS core/shell QDs emitting around 655 nm were used in our studies. 20 μL of 1 μM (20 pmol) QDs solution were injected subcutaneously in the anterior paw of healthy nude mice and the axillary lymph node (ALN) was identified visually after injection of a blue dye. In vivo fluorescence spectroscopy was performed on ALN before the mice were sacrificed at 5, 15, 30, 60 min and 24 h after QDs injection. ALN and all other organs were removed, cryosectioned and observed in fluorescence microscopy. The organs were then chemically made soluble to extract QDs. Plasmatic, urinary and fecal fluorescence levels were measured.
QDs were detected in ALN as soon as 5 min and up to 24 h after the injection. The maximum amount of QDs in the ALN was detected 60 min after the injection and corresponds to 2.42% of the injected dose. Most of the injected QDs remained at the injection site. No QDs were detected in other tissues, plasma, urine and feces.
Effective and rapid (few minutes) detection of sentinel lymph node using fluorescent imaging of quantum dots was demonstrated. This work was done using very low doses of injected QDs and the detection was done using a minimally invasive method.
PMCID: PMC2375898  PMID: 18430208
19.  Features of the popliteal lymph nodes seen on musculoskeletal MRI in a Western population 
Skeletal Radiology  2011;40(8):1041-1045.
To asses the features and explore the clinical relevance of popliteal lymph nodes (PLNs) detected on MRI examination for different pathologies of the knee.
Materials and methods
A total of 150 knee MRIs, which were conducted for various indications, were retrospectively collected from the Picture Archiving and Communication System. Imaging planes in at least two orthogonal planes were mandatory, with a field of view extending 15 cm cranial from the joint space. The localization of the PLN was determined by measuring the distance of the lowest border of the PLN to the lowest border of the lateral femoral condyle. Clinical diagnosis was obtained from radiology reports and a statistician performed the statistical analysis.
The patients were 70 males [mean age 36.6 years (range: 5–72 years)] and 80 females [mean age 41.1 years (range: 9–76 years)]. In 36.7% of the patients, a PLN was visible. The number of PLNs was negatively associated with age (p < 0.001). The mean number of PLNs was 0.5 PLN per patient. The mean length, height, and width were respectively: 0.57 cm (SD = 0.15), 0.84 cm (SD = 0.26), and 0.71 cm (SD = 0.23). The mean location was 5.8 cm (SD = 1.61). No association was found between the presence of PLNs and internal derangement, inflammation, or cancer (p = 0.368).
PLNs appearance is age related, with a higher frequency at a young age. The presence of the PLNs showed no relation to a specific clinical situation.
PMCID: PMC3125512  PMID: 21249495
Knee; Popliteal lymph node; Western population; Magnetic resonance imaging; Popliteal fossa
20.  Lymph Node Mapping in the Mouse 
Journal of immunological methods  2007;332(1-2):170-174.
Accurate identification of lymph nodes in the mouse is critical for studies of tumor metastasis, and of regional immune responses following immunization. However, these small lymphatic organs are often difficult to identify in mice using standard dissection techniques, so that larger rats have been used to characterize rodent lymphatic drainage. We developed techniques injecting dye into the mouse footpad or tail, to label the lymphatic drainage of the hind leg and flank, pelvic viscera, prostate and mammary glands. While lymphatic drainage patterns were similar in mice and rats, the inguinal lymph nodes showed distinct differences in afferent and efferent drainage. These techniques allow accurate and rapid identification of lymph nodes and lymphatic drainage in normal as well as diseased mice.
PMCID: PMC2342937  PMID: 18164026
mouse; lymph node; lymphatic drainage; Evans Blue; lymphography
21.  Lymphatic Vessel Function and Lymphatic Growth Factor Secretion after Microvascular Lymph Node Transfer in Lymphedema Patients 
Recent reports have shown that microvascular lymph node transfer may improve lymphatic drainage in lymphedema patients. Lymphatic anastomoses are expected to form spontaneously in response to lymphatic growth factor [vascular endothelial growth factor C (VEGF-C)] secreted by the transferred lymph nodes.
We have analyzed the results of 19 lymph node transfer patients operated on 2007–2012. Postoperat ive lymphatic function of the affected arm was evaluated using semiquantitative lymphoscintigraphy (transport index) and limb circumference measurements. To investigate the postoperative VEGF-C secretion, we examined axillary seroma fluid samples after different surgical operations, including lymph node transfer.
The transport index was improved postoperatively in 7 of 19 patients. Ten of the 19 patients were able to reduce or even discontinue using compression garments. Arm circumferences were reduced in 12 of 19 patients. Six of the 7 patients with preoperative erysipelas infections have not had infectious episodes postoperatively during 15–67 months follow-up. Neuropathic pain was relieved in 5 of 5 patients. VEGF-C protein was detected in the axillary seroma fluid both after lymph node transfer and normal breast reconstruction.
Reconstructing the lymphatic anatomy of the axilla with a lymph node flap may offer possibilities that other reconstructive options are lacking. However, we will need further reports and comparative studies about the clinical efficacy of this new promising technique. In addition to the transferred lymph nodes, lymphatic growth factor production may also be induced by other factors related to microvascular breast reconstruction.
PMCID: PMC4184049  PMID: 25289206
22.  The Cc Chemokine Thymus-Derived Chemotactic Agent 4 (Tca-4, Secondary Lymphoid Tissue Chemokine, 6ckine, Exodus-2) Triggers Lymphocyte Function–Associated Antigen 1–Mediated Arrest of Rolling T Lymphocytes in Peripheral Lymph Node High Endothelial Venules 
T cell homing to peripheral lymph nodes (PLNs) is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial tethering and rolling via L-selectin, firm adhesion of T cells requires rapid upregulation of lymphocyte function–associated antigen 1 (LFA-1) adhesiveness by a previously unknown pathway that activates a Gαi-linked receptor. Here, we used intravital microscopy of murine PLNs to study the role of thymus-derived chemotactic agent (TCA)-4 (secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) in homing of adoptively transferred T cells from T-GFP mice, a transgenic strain that expresses green fluorescent protein (GFP) selectively in naive T lymphocytes (TGFP cells). TCA-4 was constitutively presented on the luminal surface of HEVs, where it was required for LFA-1 activation on rolling TGFP cells. Desensitization of the TCA-4 receptor, CC chemokine receptor 7 (CCR7), blocked TGFP cell adherence in wild-type HEVs, whereas desensitization to stromal cell–derived factor (SDF)-1α (the ligand for CXC chemokine receptor 4 [CXCR4]) did not affect TGFP cell behavior. TCA-4 protein was not detected on the luminal surface of PLN HEVs in plt/plt mice, which have a congenital defect in T cell homing to PLNs. Accordingly, TGFP cells rolled but did not arrest in plt/plt HEVs. When TCA-4 was injected intracutaneously into plt/plt mice, the chemokine entered afferent lymph vessels and accumulated in draining PLNs. 2 h after intracutaneous injection, luminal presentation of TCA-4 was detectable in a subset of HEVs, and LFA-1–mediated TGFP cell adhesion was restored in these vessels. We conclude that TCA-4 is both required and sufficient for LFA-1 activation on rolling T cells in PLN HEVs. This study also highlights a hitherto undocumented role for chemokines contained in afferent lymph, which may modulate leukocyte recruitment in draining PLNs.
PMCID: PMC2195804  PMID: 10620605
homing; intravital microscopy; adhesion; T cell
23.  Inflammatory Manifestations of Experimental Lymphatic Insufficiency 
PLoS Medicine  2006;3(7):e254.
Sustained lymph stagnation engenders a pathological response that is complex and not well characterized. Tissue inflammation in lymphedema may reflect either an active or passive consequence of impaired immune traffic.
Methods and Findings
We studied an experimental model of acute post-surgical lymphedema in the tails of female hairless, immunocompetent SKH-1 mice. We performed in vivo imaging of impaired immune traffic in experimental, murine acquired lymphatic insufficiency. We demonstrated impaired mobilization of immunocompetent cells from the lymphedematous region. These findings correlated with histopathological alterations and large-scale transcriptional profiling results. We found intense inflammatory changes in the dermis and the subdermis. The molecular pattern in the RNA extracted from the whole tissue was dominated by the upregulation of genes related to acute inflammation, immune response, complement activation, wound healing, fibrosis, and oxidative stress response.
We have characterized a mouse model of acute, acquired lymphedema using in vivo functional imaging and histopathological correlation. The model closely simulates the volume response, histopathology, and lymphoscintigraphic characteristics of human acquired lymphedema, and the response is accompanied by an increase in the number and size of microlymphatic structures in the lymphedematous cutaneous tissues. Molecular characterization through clustering of genes with known functions provides insights into processes and signaling pathways that compose the acute tissue response to lymph stagnation. Further study of genes identified through this effort will continue to elucidate the molecular mechanisms and lead to potential therapeutic strategies for lymphatic vascular insufficiency.
Editors' Summary
Lymphedema is the term used to describe the swelling that can occur after surgery, especially after axillary lymph node dissection for breast cancer, when the lymph vessels that carry protein-rich interstitial fluid from the tissues to the heart are damaged. Lymphedema can be extremely unpleasant and is very hard to treat; treatments that are currently used include those aimed to help massage the flow of lymph back to the chest. Lymphedema seems to be more than just accumulation of lymph, however, as changes also occur in the tissue surrounding the damaged lymph vessels. Currently, very little is known at the most basic level about what exactly these changes are, although they appear to be similar to an inflammatory process. One way of studying such a disease process in humans is to make an animal model that mimics the human condition and study the changes that occur there. For lymphedema, such a model can be made in the mouse tail by cutting lymph vessels there.
Why Was This Study Done?
The authors wanted to look closely at what happens in the tissues surrounding damaged lymph vessels to try to understand better what these changes are. They also wanted to study the movement of the cells that are normally carried in the lymph.
What Did the Researchers Do and Find?
The mouse model that the authors developed closely simulates the characteristics of human acquired lymphedema. In the mouse tails that had had their lymph vessels damaged, the authors were able to show that the tails were swollen compared with those of normal animals and of animals that hadhad sham (pretend) surgery. In the animals with lymphedema, many small lymph vessels were seen, as the lymph was unable to flow away normally. The area affected by the lymphedema had the appearance often seen in inflamed tissue, and analysis of genes from the same area to see how active, or “expressed,” they were showed changes that are often seen in, for example, acute inflammation and wound healing. The authors also showed that when these animals were injected with immune cells marked with a light marker, they were less able to remove the cells from the circulation.
What Do These Findings Mean?
These results show that the response to lymph stagnation is complex, but looks similar to that seen in acute inflammation. These results and this model may be useful in suggesting, and at a later date perhaps testing, treatments for lymphedema. One difference, however, between this mouse model and the condition in humans is that whereas lymphedema in humans is a rather chronic condition, here the researchers were only able to look at the changes over a short period of time. In a related Perspective article, Peter Carmeliet and colleagues further discuss the clinical relevance of these findings (
Additional Information.
Please access these Web sites via the online version of this summary at
• The National Cancer Institute has information for patients and health professionals on lymphedema
• Cancerbackup is a United Kingdom cancer information service with information on many aspects of cancer, including lymphedema
• The Lymphatic Research Foundation Description has information on lymphatic system research
Rockson and colleagues characterized a mouse model of acute, acquired lymphedema using in vivo functional imaging, histopathological examination, and transcriptional profiling.
PMCID: PMC1502157  PMID: 16834456
24.  The allergy adjuvant effect of particles – genetic factors influence antibody and cytokine responses 
BMC Immunology  2005;6:11.
There is increasing epidemiological and experimental evidence for an aggravating effect of particulate air pollution on asthma and allergic symptoms and, to a lesser extent, on allergic sensitization. Genetic factors appear to influence not only the magnitude, but also the quality of the adjuvant effect of particles with respect to allergen-specific IgE (Th2-associated) and IgG2a (Th1-associated) responses. In the present study, we aimed to investigate how the genetic background influences the responses to the allergen and particles alone and in combination. We examined how polystyrene particles (PSP) affected the IgE and IgG2a responses against the model allergen ovalbumin (OVA), after subcutaneous injection into the footpad of BALB/cA, BALB/cJ, NIH and C3H/HeN mice, Further, ex vivo IL-4, IFN-γ and IL-10 cytokine secretion by Con A-stimulated cells from the draining popliteal lymph node (PLN) five days after injection of OVA and PSP separately or in combination was determined.
PSP injected with OVA increased the levels of OVA-specific IgE antibodies in all strains examined. In contrast, the IgG2a levels were significantly increased only in NIH and C3H/HeN mice. PSP in the presence of OVA increased cell numbers and IL-4, IL-10 and IFN-γ levels in BALB/cA, NIH and C3H/HeN mice, with the exception of IFN-γ in NIH mice. However, each mouse strain had their unique pattern of response to OVA+PSP, OVA and PSP, and also their unique background cytokine response (i.e. the cytokine response in cells from mice injected with buffer only).
Genetic factors (i.e. the strain of mice) influenced the susceptibility to the adjuvant effect of PSP on both secondary antibody responses and primary cellular responses in the lymph node, as well as the cellular responses to both OVA and PSP given separately. Interestingly, PSP alone induced cytokine responses in the lymph node in some of the mouse strains. Furthermore, we found that the ex vivo cytokine patterns did not predict the in vivo Th2- and Th1-associated antibody response patterns in the different mouse strains. The results indicate that insoluble particles act by increasing the inherent response to the allergen, and that the genetic background may determine whether an additional Th1-associated component is added to the response.
PMCID: PMC1182367  PMID: 15967044
25.  T cells that are naturally tolerant to cartilage-derived type II collagen are involved in the development of collagen-induced arthritis 
Arthritis Research  2000;2(4):315-326.
The immunodominant T-cell epitope that is involved in collagen-induced arthritis (CIA) is the glycosylated type II collagen (CII) peptide 256-270. In CII transgenic mice, which express the immunodominant CII 256-270 epitope in cartilage, the CII-specific T cells are characterized by a partially tolerant state with low proliferative activity in vitro, but with maintained effector functions, such as IFN-γ secretion and ability to provide B cell help. These mice were still susceptible to CIA. The response was mainly directed to the glycosylated form of the CII 256-270 peptide, rather than to the nonglycosylated peptide. Tolerance induction was rapid; transferred T cells encountered CII within a few days. CII immunization several weeks after thymectomy of the mice did not change their susceptibility to arthritis or the induction of partial T-cell tolerance, excluding a role for recent thymic emigrants. Thus, partially tolerant CII autoreactive T cells are maintained and are crucial for the development of CIA.
A discussion is ongoing regarding the possible role of cartilage-directed autoimmunity as a part of the pathogenesis of rheumatoid arthritis (RA). One possibility is that the association of RA with shared epitope-expressing DR molecules reflects a role for major histocompatibility complex (MHC) class II molecules as peptide receptors, and that the predilection of the inflammatory attack for the joint indicates a role for cartilage as a source of the antigenic peptides. A direct role for CII in the development of arthritis is apparent in the CIA model, in which a definite role for MHC class II molecules and a role for CII-derived peptides have been demonstrated [1,2,3]. Remarkably, it was found that the identified MHC class II molecule in the CIA model Aq has a structurally similar peptide binding pocket to that of the shared epitope, expressing DR4 molecules [4]. In fact, DR4 (DRB1*0401) and DR1 (DRB1*0101) transgenic mice are susceptible to CIA because of an immune response to a peptide that is almost identical to that which is involved in Aq-expressing mice [5,6]. They are both derived from position 260-273 of the CII molecule; the peptide binds to the Aqmolecule with isoleucine 260 in the P1 pocket, but with phenylalanine 263 in the P1 pocket of the DR4 and DR1 molecules.
Although these findings do not prove a role for CII in RA, they show that such recognition is possible and that there are structural similarities when comparing mouse with human. However, there are also strong arguments against such a possibility. First, arthritis can evolve without evidence for a cartilage-specific autoimmunity, as seen with various adjuvant-induced arthritis models [7,8] and in several observations using transgenic animals with aberrant immunity to ubiquitously expressed proteins [9,10,11]. Moreover, the MHC association in the adjuvant arthritis models correlates with severity of the disease rather than susceptibility [7,8], as has also been observed in RA [12]. Second, it has not been possible to identify the CII-reactive T cells from RA joints, or to achieve a strong and significant CII proliferative response from T cells derived from RA joints. Most recently these negative observations were corroborated using DR4+CII peptide tetramer reagents [13]. On the other hand, it has also been difficult to isolate autoreactive CII-specific T cells from CIA, and it can be anticipated that, even in the CIA model, T cells that are specific for CII will be hard to find in the joints [4].
We believe that the explanations for these observations in both experimental animals and humans are related to tolerance. The CIA model in the mouse is usually induced with heterologous CII, and is critically dependent on an immune response to the glycosylated CII peptide 256-270, which is bound to the MHC class II Aq molecule. In CII transgenic mice, expressing the heterologous (rat) form of the immunodominant CII 256-270 epitope in cartilage, we observed partial T-cell tolerance. This tolerance is characterized by a low proliferative activity, but with maintained effector functions such as production of IFN-γ and the ability to give help to B cells to produce anti-CII IgG antibodies [14]. Interestingly, these mice were susceptible to arthritis. However, a possibility was that T cells that had newly emerged from the thymus and that were not yet tolerized when the mice were immunized with CII led to the induction of arthritis. We have now addressed this possibility and found that induction of tolerance occurs within a few days, and that mice lacking recent thymic emigrants (ie thymectomized mice) display partially tolerant T cells and susceptibility to arthritis to the same extent as nonthymectomized mice. In addition we found that T cells that are reactive with the nonmodified peptides are relatively more affected by tolerance than T cells that are reactive with the more immunodominant glycosylated variants.
To investigate the possibility that T cells that are naturally tolerant to the cartilage protein CII are involved in the development of arthritis, and to exclude a role for nontolerized recent thymic T-cell emigrants in the development of arthritis.
Materials and methods:
A mutated mouse CII, expressing glutamic acid instead of aspartic acid at position 266, was expressed in a transgenic mouse called MMC (mutated mouse collagen) that has been described earlier [14]. The mice were thymectomized, or sham-operated, at 7 weeks of age and allowed to recover for 4 weeks before being immunized with rat CII in complete Freund's adjuvant. Arthritis development was recorded and sera analyzed for anti-CII IgG, IgG1 and IgG2a levels. To assay T-cell effector functions, other MMC and control mice were immunized in the hind footpads with rat CII in complete Freund's adjuvant, and the draining popliteal lymph nodes were taken 10 days later. The lymph node cells (LNCs) were used for proliferation assay, IFN-γ enzyme-linked immunosorbent assay (ELISA) and B-cell enzyme-linked immunospot (ELISPOT). For the proliferation assay, 106 cells were put in triplicate cultures in microtitre wells together with antigen and incubated for 72h before thymidine-labelling and harvesting 15-18h later. For IFN-γ ELISA analysis, supernatant from the proliferation plates was removed before harvesting and used in an ELISA to quantify the amount of IFN-γ produced [15]. B-cell ELISPOT was performed to enumerate the number of cells producing anti-CII IgG [16].
T-cell lines that were reactive towards rat CII were established by immunization with rat CII. An established T-cell line that was reactive with CII and specific for the CII 256-270 peptide was restimulated with freshly collected, irradiated, syngenic spleen cells and rat CII for 3 days followed by 2 weeks of IL-2 containing medium. Immediately before transfer, the cells were labelled with the cytoplasmic dye 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) [17]. Labelled cells (107) were injected intravenously into transgenic MMC mice and nontransgenic littermates. The mice were killed 4 days after cell transfer, and the concentration of CFSE-labelled cells was determined by flow cytometry.
Results and discussion:
To investigate whether and how quickly CII-reactive T cells will encounter CII in vivo, an established T-cell line that is reactive towards rat CII was labelled with the cytoplasmic dye CFSE and transferred into MMC-QD and control mice. Four days later the mice were killed, and it was found that MMC-transgenic mice had dramatically fewer CFSE-labelled cells in the spleen than did nontransgenic littermates (0.11% compared with 0.57%). Similarly, reduced numbers of CFSE-positive cells were observed in blood. This indicates that the T cells encountered the mutated CII that was present in the cartilage of MMC mice, but not in the nontransgenic littermates. Presumably, CII from cartilage is spread by antigen-presenting cells (APCs) to peripheral lymphoid organs. This observation also suggests that newly exported T cells from the thymus will be tolerized to CII in the periphery within less than 4 days.
To further investigate whether the MMC mice harbours naïve or tolerized T cells, the mice were immunized with CII at different time points after thymectomy that were well in excess of the times required for their encounter with CII. After 10 days, the response was analyzed in vitro towards both the nonglycosylated and the glycosylated CII 256-270 peptides as well as towards purified protein derivative. The galactosylated form of the peptide (Fig. 1) was used because this is the most immunodominant modification [18]. In contrast to control mice, LNCs from transgenic mice did not proliferate significantly towards the nonglycosylated peptide, indicating that these cells have been specifically tolerized, which is in accordance with earlier observations [14]. A reduced, but still significant proliferation was also observed toward the immunodominant glycosylated CII peptide. Most important, however, was that the proliferative response in the MMC mice did not decrease after thymectomy. Similarly, a significant IFN-γ production towards the glycosylated CII peptide was observed in the MMC mice. The response was somewhat reduced compared with that observed in nontransgenic littermates, and this was especially true for the response toward the nonglycosylated peptide. Again, no decrease in the MMC response by thymectomy was observed. Taken together, the T-cell response in transgenic mice was reduced in comparison with that in the nontransgenic littermates. Furthermore, the response in transgenic animals did not decrease by thymectomy (4 or 8 weeks before immunization), showing that autoreactive T cells are still maintained (and partially tolerized) with significant effector functions at least up to 8 weeks after thymectomy, excluding a exclusive role for recent thymic emigrants in the autoimmune response towards CII. To investigate whether thymectomized mice, lacking recent CII-specific thymic emigrants, were susceptible to CIA, mice were immunized with CII 4 weeks after thymectomy and were observed for arthritis development during the following 10 weeks. Clearly, the thymectomized MMC mice were susceptible to arthritis (five out of 18 developed arthritis; Fig. 2), and no significant differences in susceptibility between thymectomized and sham-operated mice, or between males and females, were seen. In accordance with earlier results [14], MMC transgenic mice had a significantly reduced susceptibility to arthritis as compared with the nontransgenic littermates (P < 0.0001 for arthritic scores, disease onset and incidence). All mice were bled at 35 days after immunization, and the total levels of anti-CII IgG were determined. Transgenic mice developed levels of anti-CII IgG significantly above background, but the antibody titres were lower than in nontransgenic littermates (P < 0.0001). No effect on the antibody levels by thymectomy was observed, nor did thethymectomy affect the distribution of IgG1 versus IgG2a titres,indicating that the observed tolerance is not associated with a shift from a T-helper-1- to a T-helper-2-like immune response. These findings show that T cells that are specific for a tissue-specific matrix protein, CII, are partially tolerized within a few days after thymus export and that these tolerized cells are maintained after thymectomy. Most important, mice that lack newly exported CII reactive T cells are still susceptible to CIA, suggesting that the partially tolerant T cells are involved in development of arthritis.
In the light of these data it is possible to explain some of the findings in RA. T-cell reactivity to CII has been shown in RA patients, but with a very weak proliferative activity [19,20]. This is fully compatible with observations in mouse and rat CIA when autologous CII, and not heterologous CII, are used for immunization. This is particularly true if the responses are recorded during the chronic phase of disease, in which the antigen-specific T-cell responses seem to be suppressed in both humans and experimental animals. These observations were confirmed in a recent report [21] in which it was shown that CII-reactive T-cell activity could be detected in RA patients if IFN-γ production but not proliferation was measured. In the present studies in mice the strongest response is seen towards post-translational modifications of the peptide. Because the T-cell contact points are the same whether the peptide is bound to DR4 or to Aq, it is fully possible that post-translational modifications of the peptide also plays a significant role in humans [22]. The fact that IgG antibodies specific for CII are found in many RA patients could be explained by maintained B-cell helper functions of CII-reactive T cells. In fact, it has been reported [23,24] that the occurrence of IgG antibodies to CII is associated with shared epitope DR4 molecules. These observations are thus compatible with a role for CII reactivity in RA. To avoid any confusion, it needs to be stressed that RA is a heterogeneous syndrome in which not only CII, but also other cartilage proteins and other mechanisms are of importance. Such a pathogenic heterogeneity is reflected by the multitude of experimental animal models that have demonstrated how many different pathways may lead to arthritis [25].
PMCID: PMC17814  PMID: 11056672
autoimmunity; rheumatoid arthritis; T lymphocytes; tolerance; transgenic

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