Inhibition of formation of synapses between dendritic cells and lymphocytes in skin lymph in an allogeneic reaction by cyclosporine and tacrolimus.

Skin, an important component of composite tissue allografts is considered to be among the most immunogenic of tissues. The mechanisms of resistance of skin allografts to pharmacological immunosuppression remain unknown. We investigated this problem at the level of antigen presentation by graft dendritic cells (DC) to recipient lymphocytes (L). Cells obtained from lymph draining skin were examined for formation of synapses, necessary for antigen presentation, in the presence of cyclosporine (CsA) or tacrolimus (FK 506). In culture the frequency of DC-L synapses was greater in allogeneic than syngeneic combinations. Cells treated with FK 50% showed a decreased rate of formation of autologous or allogeneic DC-L synapses and lower expression of CD49d. The suppressive effect of FK 506 on DC-L synapse formation may explain the effectiveness of this drug for skin allograft survival.

Formation of synapses between dendritic cells and lymphocytes in skin lymph in an allogeneic reaction.

Skin is an important component of a composite tissue allograft and is considered among the most immunogenic of tissues. Because of the skin's high degree of immunogenicity, until recently it was widely assumed that the dosage of immunosuppressive drugs required to prevent rejection was too high to be used safely in the clinical setting. The mechanism of resistance of skin allografts to pharmacological immunosuppression remains unknown. We investigated this problem at the level of antigen presentation by graft dendritic cells to recipient lymphocytes. Cells obtained from lymph draining skin were used and formation of synapses, necessary for antigen presentation, in the presence of CsA or FK506 was studied. The study provided the following information: a) increased frequency of allogeneic compared to syngeneic DC-L synapses in culture, b) increased expression of CD49d on lymph allogeneic DC and L and of HLA DR on L forming synapses, c) lack of a downregulating effect of CsA on the allogeneic DC-L synapse formation rate, d) decreased formation rate of autologous and allogeneic DC-L synapses and lower expression of CD49d and HLA DR of cells treated with FK506. The suppressive effect of FK506 on DC-L synapses formation may explain the mechanism of effectiveness of this drug on skin allograft survival.

Skin allografts--lymph veiled (dendritic) cells are responsible for initiation of rejection in canine skin/SCID mouse chimera model.

Skin allografts are acutely rejected despite of intensive immunosuppressive therapy. Resistance of skin dendritic cells to immunosuppressive drugs and irradiation may be responsible for this phenomenon. Skin allograft is a site of interaction between the dendritic cells and lymphocytes of the donor and host origin and "direct" and "indirect" pathway of antigen presentation. Increasing evidence supports the significant role for the "indirect" allorecognition in graft rejection. To investigate a critical role of skin dendritic cells in the "indirect" allorecognition and graft destruction we have used a canine skin to severe-combined-immunodeficient (SCID)-mice transplantation model. At the time the skin grafts were deprived of own dendritic (Langerhans) cells, SCID mice were reconstituted with allogeneic canine whole lymph leukocytes, lymph lymphocytes, lymph veiled (dendritic) cells or peripheral blood mononuclear cells, and an early phase of skin rejection was evaluated in histopathological studies. We demonstrated that circulating canine allogeneic veiled cells facilitate recruitment of T lymphocytes into skin graft and promote an extensive graft destruction, compared to the less expressed effect of allogeneic peripheral lymph lymphocytes or blood mononuclear cells. These drug and radiation-resistant dendritic cells may be responsible for initiation of the difficult to control rejection process.

Pathways of antigen traffic from skin allotransplant to recipient lymph nodes--an evolutionarily developed route for initiation of rejection of foreign antigens.

Immune events developing in the bed of skin allograft and draining lymphatics and lymph nodes are probably a copy of what happens in skin after invasion by bacteria, viruses or fungi. The mechanism of local immune response developed in skin during the evolution and is highly conserved and efficient in elimination of foreign antigens. This is why the take of a skin allograft is so difficult and immunosuppressive measures applied after allogeneic skin transplantation remain so ineffective. Authors present their results of studies on the human skin immune humoral and cellular factors, underlining their specificities and differences compared with blood. They also analyze the role of non-immunological factors, such as tissue fluid formation and lymph flow in transportation of alloantigen to the lymph nodes. The migratory properties of immune cells are an indispensable factor in transfer of alloantigen to the lymph nodes. Understanding of the evolutionarily developed immune events in skin may allow to analyze the process of skin allograft rejection and can give hints for more effective immunosuppressive policy.

Reactivity of antibodies directed against human antigens with surface markers on canine leukocytes.

A panel of anti-human antibodies was used in order to investigate their cross-reactivity with canine leukocytes. The labeling was carried out at the microscopic level by immunocytochemical staining. Of 50 antibodies 22 cross-reacted with canine leukocytes from afferent lymph and peripheral blood. All leukocytes reacted with MHM23 (CD18). Two anti-HLA DR antibodies, DK22 and L243, reacted mostly with veiled cells and with PHA-stimulated lymphocytes, whereas TAL1B5 was cross-reactive with all canine lymphocytes. The activation markers CD25, Ki-67, PCNA were identified on PHA-stimulated lymphocytes with ACT1, Ki-67 and PC10 clone produced antibodies. Canine eosinophils reacted with MHM6 (CD23) antibody. A large number of antibodies reacted with canine lymph veiled cells. Canine granulocytes and a subset of lymphocytes were stained with the anti-CD15 antibody only after treatment of cytospins with neuraminidase.

Cytokines and adherence molecules involved in spontaneous dendritic cell-lymphocyte clustering in skin afferent lymph.

The skin afferent lymph dendritic cell (DC) spontaneously forms clusters with autologous T cells. The role of adhesion molecules and cytokines in this process was investigated. Analysis of the expression of adhesion receptors on the canine peripheral lymph DC revealed the presence of CD54, CD58, CD18 as well as CD49d and CD49e molecules and cell surface fibronectin. The CD54 and CD58 molecules were found to play a key role in the 'spontaneous' lymph cell clustering. Antibody against fibronectin, a substrate for CD49d and CD49e receptors, reduced DC-lymphocyte binding. Analysis of the effect of cytokines revealed that the pro-inflammatory IL1 beta rather than IL1 alpha, and TNF alpha may be responsible for the enhanced lymph cell in vitro clustering. The IL6 had no such augmenting effect. The enhancing effect of endogenous IL1 beta present in lymph was reduced by the IL1 beta neutralizing antibody. The effect of exogenously added IL1 beta was also limited by the IL1 receptor antagonist. The IL1Ra alone had no effect on cell binding, even when used in the high doses. Neutralizing of IL1Ra in lymph with the specific antibody brought about augmented cluster formation. The enhancing properties of TNF alpha on cell binding were reduced by the TNF alpha neutralizing antibody. The IL10 significantly limited lymph DC cluster formation with T cells. In conclusion, these data demonstrate that the present in lymph IL1 beta and TNF alpha may be responsible for the observed in vitro enhanced cluster formation of lymph DC with autologous T lymphocytes. Cell binding can be reduced by IL1Ra and by IL10. It provides insight into the potential clinical use of these inhibitors.

Immune events in skin. II. Inhibition of dendritic (veiled) cells function and traffic with anti-lymph dendritic cell sera.

The migrating dendritic cells (DC) from the canine skin-draining afferent lymph are the most potent stimulators of the allogeneic MLR of all other lymph cell subpopulations. The MLR could be inhibited by addition of xenogeneic (rabbit), polyclonal anti-lymph dendritic cell serum (ADCS) to the cultures. ADCS blocked the presentation of Ia and CD1 antigens on the surface of DC. Studies using mouse anti-human monoclonal antibodies, cross-reactive with canine cells, indicate that blocking of Ia, but not of CD1 antigen, is responsible for inhibition of MLR. ADCS abrogated also the accessory function of DC in the lymph lymphocytes response to PHA. The inhibition reached 73%, when the serum was added for the whole period of the culture, and 23% after preincubation of DC with ADCS (anti-dendritic cell serum). ADCS administered subcutaneously into the dorsal area of hind paws caused a selective, transient disappearance of DC from the afferent lymph of normal dogs, which lasted for 2 h after the injection. We conclude, that the functional and physical elimination of migrating skin DC mediated by ADCS in vitro and in vivo, justify the possible use of this serum in the combined immunotherapy directed at the prolongation of skin graft survival.

Spontaneous cluster formation of dendritic (veiled) cells and lymphocytes from skin lymph obtained from dogs with chronic lymphedema.

To examine the mechanism of spontaneous attachment of afferent lymph lymphocytes to dendritic cells (veiled), we sampled and tested cells from skin lymph in 3 dogs with chronic lymphedema. There were 3.3 +/- 2.8% veiled cells in clusters with lymphocytes in lymph obtained directly from dilated dermal lymphatics by percutaneous puncture. The number of ex vivo clusters forming in the collected lymph samples increased as a function of time and was temperature dependent. The ability of veiled cells to bind lymphocytes was independent of divalent cations but reduced by xylocaine and retinoic acid. Among various steroids tested only methylprednisolone showed an inhibitory effect on cluster formation. Indomethacin and acetylsalicylic acid had no blocking activity on cell binding. Moreover, no effect was seen by cyclosporin A and azathioprine. However, FK 506 had a potent inhibitory effect on spontaneous cluster formation. This study suggests that cluster formation by skin lymph veiled cells and lymphocytes is a spontaneous process which can be controlled in vitro.

Development of antiserum against veiled (dendritic) cells of canine afferent lymph: implications for transplantation biology.

A method is described for production of anti-veiled cell serum against veiled cells (VC) or dendritic cells obtained from canine skin lymph. By use of discontinuous Percoll gradient, VC from lymph were enriched to about 50% of the entire lymph cell population. After immunization of rabbits with the priming total dose of 10(7) VC (intramuscular, subcutaneous, intracutaneous) and with the same total booster injection (intravenous), the sera obtained were cytotoxic mainly for VC, with cytotoxin titer 1:16-1:32 and for agglutinin 1:256-1:512, respectively. Antisera used in vitro blocked the Ia and CD1 antigens of VC on smears and inhibited the accessory function of VC in cell response to phytohemagglutinin (PHA) and their stimulatory activity in mixed leukocyte reaction (MLR). In vivo, the local, intracutaneous administration of antisera led to transient depletion of VC from afferent lymph, and to reduction of mononuclear cells in the T-dependent areas in regional lymph nodes.

Immune events in skin. I. Spontaneous cluster formation of dendritic (veiled) cells and lymphocytes from skin lymph.

To investigate the mechanism of spontaneous attachment of afferent lymph lymphocytes to dendritic cells, cells from canine skin lymph were used. There were 3.3 +/- 2.8% of veiled cells in clusters found in lymph flowing from the cannulated lymph vessel. The number of clusters forming ex vivo in the collected lymph samples increased as a function of time and was temperature dependent. Incubation of cells with proteolytic enzymes or monosaccharides did not alter cell interactions. The ability of veiled cells to bind lymphocytes was independent of divalent cations but reduced by xylocaine and retinoic acid. Among steroids only methylprednisolone showed an inhibitory effect on cluster formation. Indomethacin and acetylsalicylic acid had no blocking activity on cell binding. Also, no effect was seen after treatment with cyclosporine A and azathioprine. An enhanced cluster formation after desialation with neuraminidase was observed. The desialated cells were cultured in order to study their stimulatory and accessory cell functions. No enhancement of autologous mixed leucocyte reaction was seen, but a significantly higher responsiveness to a suboptimal dose of phytohaemagglutinin was observed. The N-ase-mediated non-specific cell attachment could be abrogated by cell washing or treatment with EDTA or xylocaine. This study indicates that cluster formation by skin lymph veiled cells and lymphocytes is a spontaneous process which cannot be controlled by means usually effective in regulating the in vitro induced clustering of antigen-stimulated cells.

Effect of acute venous hypertension on erythrocyte, leukocyte, and plasma protein extravasation in the dog hindlimb.

The effect of acute venous hypertension on the extravasation of plasma proteins, erythrocytes, and leukocytes into regional lymph was studied in the dog hindlimb. Although the lymph protein transport sharply rose with hindlimb phlebohypertension, capillary permeability was unchanged with retention in draining lymph of a normal proportion of large to small molecular weight proteins. Leukocyte transport also increased initially but then progressively decreased despite persistent venous hypertension. Lymph transport of erythrocytes was also high during venous hypertension but in contrast to white cells, it remained at that level as long as venous pressure was elevated. These findings suggest that different pathways and mechanisms are involved in the capillary extravasation of plasma proteins and cells capable of intrinsic motility (i.e., leukocytes) and those without independent motion (i.e., erythrocytes).

Functional characteristics of veiled cells from canine prenodal lymph.

The surgical interruption of afferent lymphatics in the hind limb of dog leads to peripheral lymph stasis. The stagnated lymph contains large numbers of immunocompetent cells originating solely from the skin. This experimental model allows a study of the functions of the afferent skin-draining lymph cell population, the recovery and assessment of the lymphokines and other mediators liberated by these cells during the culture, and the production of anti-sera against different types of lymph cells. In the present study, we focused on the functional, morphological and cytochemical evaluation of the non-lymphoid cells, isolated from the whole lymph cell population by means of the gradient centrifugation technique. The non-lymphoid cells were large, with an irregularly-shaped nucleus and numerous cytoplasmic projections, giving them a "veiled" cell (VC) appearance. All VC were strongly positive for DLA-class II antigens and membrane-associated ATP-ase, and 60% of them exhibited the activity of non-specific esterase. In the functional assays, VC displayed the potent accessory-cell activity in the mitogen-induced response of autologous blood- and lymph-derived lymphocytes. In the mixed leukocyte cultures, VC acted as stimulators of the allogeneic and autologous lymphocyte proliferation. The high spontaneous and mitogen-induced responsiveness of the whole lymph cell population was found to be dependent on the presence of VC. The small number of VC (5% of cultured cells) was sufficient to produce the above-mentioned effects. These results indicate that VC is a cell responsible for the antigen presentation in the skin-associated immune reactions in dog, which is relevant to the observations on similar cells from the other species.

A single step centrifugation method for the enrichment of veiled cells from canine afferent lymph.

Veiled cells (VC) present in the afferent lymph of dogs with chronic lymphoedema could be enriched from 6% to about 50% VC by density gradient centrifugation on 15% metrizamide or discontinuous Percoll gradients. The recovery of VC was about 40% from 0.22 +/- 0.07 X 10(6) VC/ml of lymph. The cells were strongly Ia positive and had cytoplasmic S 100 protein. They were also strongly ATP-ase positive and showed heterogeneity in acid phosphatase, peroxidase and non-specific esterase activity. Low density VC from canine afferent lymph were able to stimulate both blood and lymphatic lymphocytes in autologous mixed leukocyte reaction when present at concentration as low as 5% of cultured cells.

Spontaneously active suppressive cells in canine peripheral blood.

The phenomenon of the unusually high spontaneous suppressive activity of cells in peripheral blood of dogs was analysed. The m/c (mitomycin C)-treated population of peripheral blood leucocytes (PBL) contained cells able to reduce the responsiveness of autologous cells by 48 +/- 15% (P less than 0.01) and their activity was not indomethacin dependent. Thoracic duct lymphocytes (TDL) did not reduce the response of PBL to PHA, neither did cell crowding. The supernatants from 24-h cultures of m/c-treated PBL did not affect the response to PHA, and parallelly precultured cells inhibited the proliferation of PBL to a lesser degree (24 +/- 9%) than the fresh cells (50 +/- 16%, P less than 0.05). Addition of m/c-treated polymorphonuclear cells at PMN to PBL ratios of 1:4 and 1:1 progressively inhibited PBL reactivity to PHA, from 29.5 +/- 3.5% to 68.5 +/- 9%, respectively, and the supernatants from 24-h cultures of PMN reduced the proliferation by 48 +/- 2.8%. The neutrophil-derived inhibitory factor(s) was non-cytotoxic and reduced the formation of blasts to 61.5 +/- 3.5% of the control values. These results indicate that dog PBL from Lymphoprep gradient contain a population of non-recirculating, short-lived, spontaneously suppressive cells, mainly PMN, which modulate T cell reactivity in vitro, suggesting that neutrophils may be able to exert a regulatory effect in vivo.