Vascular Changes in Popliteal Lymph Nodes due to Antigen Challenge in Normal and Lethally Irradiated Mice

The microvascular system of the murine popliteal lymph node was investigated using scanning electron microscopy of microcorrosion casts. Time-dependent changes in the microvasculature following regional antigen challenge in normal and lymphocyte-depleted mice were studied. Normal lymph node microvasculature exhibited a significant increase in both the vascular bed and post-capillary venules containing high-endothelium in response to antigen challenge. Lymph nodes of lymphocyte-depleted mice showed no microvascular size increase following antigen challenge and a reduction in the amount of high-endothelium was observed

Chemoattractant Receptor-Induced Phosphorylation of L-Selectin

The selectin adhesion molecules and chemoattractant receptors synergistically regulate leukocyte migration into lymphoid tissues and sites of inflammation, but little is known about how these families of receptors modulate each other\u27s function. In this study, L-selectin was found to be phosphorylated in lymphoblastoid cell lines, and phosphorylation was enhanced by phorbol ester (phorbol 12-myristate 13-acetate (PMA)) treatment. Interactions between L-selectin and chemoattractant receptors were therefore examined using transfected rat basophilic leukemia cell lines (RBL-2H3) that expressed human L-selectin along with human leukocyte chemoattractant receptors. L-selectin was rapidly phosphorylated in cells treated with chemoattractants, thrombin, IgE receptor agonists, or PMA. Pertussis toxin or the protein kinase C inhibitor, staurosporine, completely blocked chemoattractant receptor-induced phosphorylation of L-selectin. PMA-induced phosphorylation was on serine residues within the cytoplasmic tail of L- selectin that have been well conserved during recent evolution. Although L- selectin phosphorylation was not essential for basal levels of adhesion through L-selectin in transformed cell lines, the rapid increase in ligand binding activity of L-selectin that occurs following leukocyte activation was blocked by staurosporine. These results demonstrate that L-selectin can be phosphorylated following engagement of chemoattractant receptors and suggest that this may be a physiologically relevant mechanism for the synergistic regulation of these receptors during leukocyte migration

Regulation of L-Selectin–mediated Rolling through Receptor Dimerization

L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin–GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by ∼700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors

Vascular endothelial growth factor receptor inhibitor SU5416 suppresses lymphocyte generation and immune responses in mice by increasing plasma corticosterone.

Inhibitors of vascular endothelial growth factor and its receptors (VEGFRs) are attractive therapeutic candidates for cancer treatment. One such small molecule VEGFR inhibitor, SU5416, limits angiogenesis in vivo and is widely used for investigating VEGFR signaling in tumor pathophysiology. Herein, we describe novel actions of SU5416 on the immune system. Treatment of mice with SU5416 for 3 days induced significant reductions in size and cellularity of peripheral lymph nodes. Interestingly, SU5416 did not affect initial lymphocyte localization to peripheral lymph nodes but did reduce lymphocyte accumulation during long-term migration assays. Treatment with SU5416 also induced severe loss of double-positive thymocytes resulting in thymic atrophy and a reduction in peripheral B cells. Furthermore, immune responses following immunization were reduced in mice treated with SU5416. Findings of thymic atrophy and reduced weight gain during SU5416 treatment suggested elevated corticosterone levels. Indeed, a significant 5-fold increase in serum corticosterone was found 4 hours after treatment with SU5416. Importantly, adrenalectomy negated the effects of SU5416 treatment on primary immune tissues, and partial reversal of SU5416-induced changes was observed following blockade of glucocorticoid receptors. SU5416 has been reported to inhibit the activation of latent transforming growth factor (TGF)-β, a cytokine involved in the regulation of glucocorticoid release by the adrenal glands. Interestingly, treatment with a TGF-β receptor inhibitor, showed a similar phenotype as SU5416 treatment, including elevated serum corticosterone levels and thymic atrophy. Therefore, these results suggest that SU5416 induces glucocorticoid release directly from the adrenal glands, possibly by inhibition of TGF-β activation

Tissue cell counts and frequencies in adrenalectomized mice^a.

a<p>Surgically adrenalectomized mice were treated with SU5416 (25 mg/kg/day) or equivalent amounts of vehicle. Tissues were harvested after 3 days and labeled for flow cytometric analysis. Spleen and PLN were labeled for detection of CD4, CD8, and CD19 (B cells). Thymus was labeled for CD4 and CD8. Bone marrow was labeled for B220, IgM, and IgD. *Differences between vehicle and SU5416 were significant; p<0.05.</p>b<p>Abbreviations used: DP, CD4 and CD8 double-positive; BM, bone marrow; PLN, peripheral lymph node; Pro/Pre, IgM^-IgD^– progenitor/precursor B cells; Imm, IgM⁺IgD^– immature B cells; Mature, IgM⁺IgD⁺ mature B cells.</p>c<p>Frequencies and total cell number of B cell populations in BM were calculated by gating on B220⁺ cells.</p

SU5416 treatment reduces immune responses.

<p>A) Mice were immunized unilaterally as described in Fig. 1. Mice were treated with SU5416 (25 mg/kg/day) or vehicle control starting on the day of immunization. After 3 days, mice were pulsed with BrdU for 1 hour, control and draining PLN were harvested and labeled with fluorochrome-conjugated antibodies to detect CD45 and BrdU, and analyzed by flow cytometry. Values represent the mean ± SEM percentage or number of CD45⁺ BrdU⁺ cells in each tissue from 4–5 mice per group. B) Mice were immunized in all limbs on days 0 and 28 with DNP-KLH-Alum and treated with SU5416 (50 mg/kg, twice per week) or vehicle control. Serum was collected 7, 14, 21 and 35 days following initial immunization and analyzed by isotype-specific ELISA for the presence of DNP-specific antibodies. Values represent the mean ± SEM levels of DNP-specific IgM and IgG1 antibodies from 5 mice per group. *Differences in the mean values between SU5416 and vehicle control treatments were significant; p<0.05.</p

SU5416 treatment transiently elevates serum corticosterone through inhibition of TGF-β activation.

<p>A) Mice were treated with 25 mg/kg SU5416 or vehicle control, serum was collected 4 or 24 hours later and the level of corticosterone was measured by competitive ELISA. B) Adrenalectomized mice were treated with 25 mg/kg/day SU5416 or vehicle control for 3 days. Thymocyte subsets were determined following labeling with fluorochrome-conjugated antibodies to detect CD4 and CD8 and flow cytometry analysis. Values indicate the mean ± SEM cell number of each subset from 5–6 mice per group. C) Mice were treated with 25 mg/kg/day SU5416 or vehicle in the absence or presence of 50 mg/kg/day RU486 for 3 days. Thymocyte subsets were determined as described in B. Values indicate the mean ± SEM cell number of each subset from 5–6 mice per group. D) Mice were treated with 25 mg/kg SB431542 or vehicle control for 1.5 or 4 hours and the level of corticosterone was measured as above. For both A and C, symbols represent results from individual animals and the horizontal lines indicate the mean ± SEM concentration of corticosterone in each group from 4–5 mice per group. E) Mice were treated with 25 mg/kg/day SB431542 or vehicle control for 3 days. Thymocyte subsets were determined as above. Values indicate the mean ± SEM cell number of each subset from 5–7 mice per group. *Differences in the mean values between SU5416 or SB431542 and vehicle control treatments were significant; p<0.05.</p

SU5416 treatment reduces lymphocyte accumulation in PLN.

<p>Recipient mice were immunized as described in Fig. 1. A) Mice were treated with SU5416 (25 mg/kg/day) or vehicle, or bevacizumab or Hu IgG starting on the day of immunization. Three days following immunization, donor splenocytes were biotinylated and transferred into the above treated recipient mice. One hour after transfer, control and draining PLN were harvested, and single-cell suspensions were labeled with fluorochrome-conjugated avidin to detect transferred biotinylated cells and analyzed by flow cytometry. B) Mice were treated as above but the migration of CFSE-labeled lymphocytes was analyzed 48 hours following transfer into recipient mice. C) Experiments (48 hour migration assays) were performed as described in B and further analyzed to determine lymphocyte subsets. Specifically, recipient cell suspensions were labeled with fluorochrome-conjugated antibodies to detect CD4, CD8, CD19 (B cells), and CD44 (CD44^high effector/memory cells), and analyzed by flow cytometry. Values indicate the mean ± SEM percent of adoptively transferred cells (% of transferred cells) that were recovered from each tissue from 3–4 mice per group. *Differences in the mean values between SU5416 and vehicle control treatments were significant; p<0.05.</p