Migratory capacity and function of dendritic cells from mesenteric afferent lymph nodes after feeding a single dose of vitamin A.

Lamina propria dendritic cells (DCs) have a permanent turnover with constitutive migration to mesenteric lymph nodes and replenishment by progenitors. Luminal bacteria and dietary constituents provide key signals that endow DCs their unique properties in vivo. Taking into account that the intestinal immune system is greatly influenced by retinoids, we evaluated in B6 mice 3, 8, 16 and 24 h after feeding a single dose of vitamin A phenotype and function of cells present in mesenteric afferent lymph nodes as well as signals involved in migration. We studied the frequency of CD11c+MHC-II+CD103+CD86+ and RALDH+ DCs by flow cytometry, we determined CCL-21 and D6 levels in tissue homogenates by Western blot, and we co-cultured cells isolated from afferent lymphatics with sorted CD4+ lymphocytes to assess Foxp-3 induction and homing receptor expression. Sixteen hours after vitamin A administration, DCs isolated from afferent lymphatics were able to induce homing receptors and Foxp3 expression in CD4+ lymphocytes. Our results show that a single dose of vitamin A generated a stream of signals and amplified the tolerogenic activity of DCs migrating to lymphoid tissue.

Lymphatic pump manipulation mobilizes inflammatory mediators into lymphatic circulation.

Lymph stasis can result in edema and the accumulation of particulate matter, exudates, toxins and bacteria in tissue interstitial fluid, leading to inflammation, impaired immune cell trafficking, tissue hypoxia, tissue fibrosis and a variety of diseases. Previously, we demonstrated that osteopathic lymphatic pump techniques (LPTs) significantly increased thoracic and intestinal duct lymph flow. The purpose of this study was to determine if LPT would mobilize inflammatory mediators into the lymphatic circulation. Under anesthesia, thoracic or intestinal lymph of dogs was collected at resting (pre-LPT), during four minutes of LPT, and for 10 min following LPT (post-LPT), and the lymphatic concentrations of interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-γ, tissue necrosis factor α,  monocyte chemotactic protein-1 (MCP-1), keratinocyte chemoattractant, superoxide dismutase (SOD) and nitrotyrosine (NT) were measured. LPT significantly increased MCP-1 concentrations in thoracic duct lymph. Further, LPT increased both thoracic and intestinal duct lymph flux of cytokines and chemokines as compared with their respective pre-LPT flux. In addition, LPT increased lymphatic flux of SOD and NT. Ten minutes following cessation of LPT, thoracic and intestinal lymph flux of cytokines, chemokines, NT and SOD were similar to pre-LPT, demonstrating that their flux was transient and a response to LPT. This re-distribution of inflammatory mediators during LPT may provide scientific rationale for the clinical use of LPT to enhance immunity and treat infection.

Lymphatic pump treatment augments lymphatic flux of lymphocytes in rats.

BACKGROUND: Lymphatic pump techniques (LPT) are used by osteopathic practitioners for the treatment of edema and infection; however, the mechanisms by which LPT enhances the lymphatic and immune systems are poorly understood. METHODS AND RESULTS: To measure the effect of LPT on the rat, the cisterna chyli (CC) of 10 rats were cannulated and lymph was collected during 4 min of 1) pre-LPT baseline, 2) 4 min LPT, and 3) 10 min post-LPT recovery. LPT increased significantly (p < 0.05) lymph flow from a baseline of 24 ± 5 µl/min to 89 ± 30 µl/min. The baseline CC lymphocyte flux was 0.65 ± 0.21 × 106 lymphocytes/min, and LPT increased CC lymphocyte flux to 6.10 ± 0.99 × 106 lymphocytes/min (p < 0.01). LPT had no preferential effect on any lymphocyte population, since total lymphocytes, CD4+ T cells, CD8+ T cells, and B cell numbers were similarly increased. To determine if LPT mobilized gut-associated lymphocytes into the CC lymph, gut-associated lymphocytes in the CC lymph were identified by staining CC lymphocytes for the gut homing receptor integrin α4ß7. LPT significantly increased (p < 0.01) the flux of α4ß7 positive CC lymphocytes from a baseline of 0.70 ± 0.03 × 105 lymphocytes/min to 6.50 ± 0.10 × 105 lymphocytes/min during LPT. Finally, lymphocyte flux during recovery was similar to baseline, indicating the effects of LPT are transient. CONCLUSIONS: Collectively, these results suggest that LPT may enhance immune surveillance by increasing the numbers of lymphocytes released in to lymphatic circulation, especially from the gut associated lymphoid tissue. The rat provides a useful model to further investigate the effect of LPT on the lymphatic and immune systems.

Direct lymphatic spreading route into the liver from the gallbladder: an animal experiment using pig.

In the special occasion that the physiological lymphatic flow is obstructed, gallbladder carcinoma (GBC) may spread into the liver via lymphatic route. Therefore, this study was conducted to find out the direct lymphatic route draining into the liver from the gallbladder using pigs with ligated cystic ducts. After injecting the carbon particle suspension (CH40) or the contrast medium (Lipiodol) into the subserosal layer of the gallbladder, the lymphatic route into the liver was examined both macroscopically and histologically. In controls, CH40 or Lipiodol drained along the cystic duct toward the hepatoduodenal ligament. After occlusion of cystic duct, CH40 was interrupted at the ligated point, and then spread into the liver nearby the gallbladder bed, running off to the liver hilus, toward the hepatoduodenal ligament. This route was confirmed by the Lipiodol drainage into the right median lobe of the liver, equivalent to the segments V and IV a in humans. We presented for the first time the emergence of lymphatic drainage from the gallbladder into the liver after the occlusion of physiological lymphatic route using pigs. This implies that the direct spread into the segments V and IV a of liver should be considered in the surgical treatment of advanced GBC.

Sequence analysis or rat integrin alphaE1 and alphaE2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph.

The integrin alphaOX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human alphaE that is predominantly expressed on intraepithelial lymphocytes. To clone alphaOX-62, rat probes generated using primers specific for the human alphaE sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical alpha subunits that are closely related but distinct from human alphaE were isolated. alphaE1 is predicted to be the rat homolog of mouse alphaM290 and alphaE2 corresponds to rat alphaOX-62. Immunofluorescence analysis revealed that mouse alphaE1 and rat alphaE2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where gammadelta T cells occur in lymphoid organs. Unexpectedly, in veiled cells alphaE2 is co-expressed with intracellular CD3-delta and a 33-kDa CD3 chain but not the T cell receptor. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.

Cloning of two members of the SIRP alpha family of protein tyrosine phosphatase binding proteins in cattle that are expressed on monocytes and a subpopulation of dendritic cells and which mediate binding to CD4 T cells.

Recent experimental studies have greatly clarified the function of cell surface molecules in the induction and modulation of T cell responses by antigen-presenting cells (APC). However, the differences in ability to stimulate T cells evident for different types and subpopulations of the same APC, such as dendritic cell subsets, is less well understood. This report details an investigation of an antigen expressed on monocytes that is also expressed on a subset of cattle afferent lymph veiled cells (ALVC). A cDNA library derived from cattle monocytes was screened with monoclonal antibodies (mAb) for expression in COS-7 cells. Using separate mAb for screening, two cDNA were cloned, the sequences of which showed a single long open reading frame encoding a predicted type I glycoprotein of 506 amino acids that contained three immunoglobulin superfamily domains and a long 112-amino acid cytoplasmic tail. We have termed this antigen MyD-1, reflecting its myeloid and dendritic cell distribution. Analysis of the EMBL database revealed that the molecule is a member of the recently described family of signal regulatory proteins (SIRP). The outeremost Ig domain was of the adhesion/receptor I-type, suggesting that MyD-1 might bind to a ligand on another cell. Evidence for this was subsequently obtained by demonstrating that COS-7 cells transfected with MyD-1 cDNA bound CD4 T cells and this binding was blocked by specific mAb. The potential importance of this interaction was supported by the finding that the proliferation of resting memory CD4 T cells to ovalbumin-pulsed monocytes was significantly reduced in the presence of mAb to MyD-1. A role for the molecule in the modulation of the monocyte/dendritic APC response is also predicted from the existence of multiple potential tyrosine phosphorylation sites in the cytoplasmic domain, including the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the observation that the SIRP alpha family members have been shown to bind to SHP-1 and SHP-2. Together these data indicate a possible functional importance for MyD-1 in the regulation of monocyte and dendritic cell function.

Sequence analysis of rat integrin alpha E1 and alpha E2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph.

The integrin alpha OX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human alpha E2 that is predominantly expressed on intraepithelial lymphocytes. To clone alpha OX-62, rat probes generated using primers specific for the human alpha E sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical alpha subunits that are closely related to but distinct from human alpha E were isolated. alpha E1 is predicted to be the rat homolog of mouse alpha M290 and alpha E2 corresponds to rat alpha OX-62. Immunofluorescence analysis revealed that mouse alpha E1 and rat alpha E2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where gamma delta T cells occur in lymphoid organs. Unexpectedly, alpha E2 is co-expressed with intracellular CD3-delta and a 33-kDa CD3 chain but not the T cell receptor in veiled cells. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.

[A new method for sampling lymph from animals]. / Novyi sposob zabora limfy u zhivotnykh.

In experiments conducted on locally anesthesized rats, an access to the cistern of the thoracic duct was made by the previously developed method which involved laparatomy, the exfoliation of the left kidney, arteria renalis, vena renalis, vasa lymphatica, renal lymph nodes, and ureter, followed by drawing them medially. The cistern of the thoracic duct was punctured by glass micropipettes manufactured by the Russia's patent No. 1495076, which allowed dendritic and Mott cells to be detected in central lymph.

Effect of local hyperthermia on lymph immune cells and lymphokines of normal human skin.

The effects of 3 h lasting local hyperthermia on immune cell traffic through the normal human skin to afferent lymphatics, cell phenotypes, responsiveness, and stimulatory properties were studied in eight men. Cells were harvested from lymph drained from foot skin. Heating the skin in a water bath of 44 degrees C (skin temperature 2 mm under the surface 39 degrees C) evoked an augmented traffic of mononuclear cells to lymph with preponderance of large, macrophage-like, Ia-positive cells, among them Langerhans cells. Lymphocytes obtained from the heated skin lymph revealed in cultured increased spontaneous blastic transformation rate, augmented responsiveness to phytohemagglutinin (PHA), and enhanced PHA-presenting properties to autologous peripheral blood mononuclear (PBM) cells. An increased stimulatory activity on allogeneic mixed lymphocyte reaction (MLR) was also observed. Lymph from heated skin augmented the PBM responsiveness to PHA and showed increased interleukin-1-like activity. Local heating of the skin is a potent signal initiating augmented traffic, and enhanced responsiveness and stimulatory activity of "passenger" immune cells. Their rapid nonspecific activation makes them indiscriminately active against a wide range of antigens before the specific response is developed.

Altered lymphatic circulation at the site of melanoma excision.

Local excision of malignant melanoma promotes both disruption and regeneration of regional lymphatics. These disturbances in local lymphatic drainage favor escape of residual melanoma cells either locally or in transit from more distal sites. Accordingly, a wide tridimensional resection to eradicate all local tumor and circumvent interstitial entrapment and migration of melanoma cells is still advocated. Changes in lymph vessels after excision also demand caution when distal endolymphatic isotopes are administered. Lymph leakage and trapping with overconcentration of the isotope may result in excess local irradiation and skin breakdown.

Afferent lymph and lymph borne cells: their influence on lymph node function.

In AO rats the afferent lymphatics to the right cervical lymph nodes (LN) were interrupted and the LN were encased in silicone rubber tubes to prevent reunion of the lymphatics. At regular intervals over the next 12 weeks the following were measured in comparison with the intact contralateral LN - LN weight, influx of lymphocytes from the blood, blood flow, the incorporation of 125IUdR and the incorporation of 35S-sulphate into high endothelial venules (HEV). Systematic histological observations are also reported. One day after deafferentization lymphocyte influx was significantly reduced although blood flow was unchanged and a temporary increase in LN weight was associated with crowding of the lymphatic sinuses with small lymphocytes. The subsequent decline in lymphocyte influx was biphasic and quicker than the decline of other parameters--being undetectable by 6 weeks. Flattening of HEV and diminished secretion of 35S-sulphate was noted at 1 week and progressive degeneration and eventual disappearance of the HEV network was seen by 6-12 weeks. Doubtlessly because of lack of antigenic stimulation 125IUdR incorporation, and numbers of lymphoblasts, plasma cells and finally germinal centres were progressively reduced. The numbers of macrophages and interdigitating cells (IDC) were greatly reduced by 3 weeks and very few were present at 6 weeks probably because most or all arrive in afferent lymph and have a limited life span in the LN. At 12 weeks the LN was difficult to recognize as such since only stromal cells and occasional small lymphocytes remained. In supplementary experiments u.v. irradiation of the LN at the time of deafferentization reduced lymphocyte influx without affecting blood flow suggesting that a u.v. sensitive cell like the IDC may influence lymphocyte influx. In conclusion the involution of the deafferentized LN is partly due to the lack of antigen but progression to the complete loss of specialized structure and function is probably due to lack of other factors including non-lymphoid cells that normally arrive in afferent lymph.

Pathogenesis of Salmonella-associated arithritis in the rat.

The distribution of joint lesions in rats with Salmonella-associated arthritis (SSA), as determined in a detailed survey, resembles to a great extent the pattern of small joint involvement in human rheumatoid arthritis. Such lesions, though regularly induced in the rat by the intravenous injection of live S.enteritidis, could not be evoked by the heat-killed organisms injected by various routes with and without extrinsic adjuvants. Efforts to transfer SAA from sensitized donors to either normal or primed recipients, employing lymphoid ce-ls from several sources, also failed repeatedly. Two observations, however, virtually exclude the possibility that joint damage in SAA can be the direct result of sustained intra-articular sepsis. First, the inoculation of as many as 10-3 viable inflammation. Second, the incidence of SAA was significantly lower in weanling rats in the adult controls although the growth and distribution of intravenously injected S. enteritidis was virtually identical in the two groups. Together these observations indicate that the joint damage occurring in SAA is determined by the host and not by the infecting organism. From this, it seems fair to conclude that the destructive arthritis characteristic of this syndrome is immunologically mediated.