The role for decorin in delayed-type hypersensitivity

Decorin, a small leucine-rich proteoglycan, regulates extracellular matrix organization, growth factor-mediated signaling, and cell growth. Because decorin may directly modulate immune responses, we investigated its role in a mouse model of contact allergy (oxazolone-mediated delayed-type hypersensitivity [DTH]) in decorin-deficient (Dcn(-/-)) and wild-type mice. Dcn(-/-) mice showed a reduced ear swelling 24 h after oxazolone treatment with a concurrent attenuation of leukocyte infiltration. These findings were corroborated by reduced glucose metabolism, as determined by (18)fluordeoxyglucose uptake in positron emission tomography scans. Unexpectedly, polymorphonuclear leukocyte numbers in Dcn(-/-) blood vessels were significantly increased and accompanied by large numbers of flattened leukocytes adherent to the endothelium. Intravital microscopy and flow chamber and static adhesion assays confirmed increased adhesion and reduced transmigration of Dcn(-/-) leukocytes. Circulating blood neutrophil numbers were significantly increased in Dcn(-/-) mice 24 h after DTH elicitation, but they were only moderately increased in wild-type mice. Expression of the proinflammatory cytokine TNF-α was reduced, whereas syndecan-1 and ICAM-1 were overexpressed in inflamed ears of Dcn(-/-) mice, indicating that these adhesion molecules could be responsible for increased leukocyte adhesion. Decorin treatment of endothelial cells increased tyrosine phosphorylation and reduced syndecan-1 expression. Notably, absence of syndecan-1 in a genetic background lacking decorin rescued the attenuated DTH phenotype of Dcn(-/-) mice. Collectively, these results implicated a role for decorin in mediating DTH responses by influencing polymorphonuclear leukocyte attachment to the endothelium. This occurs via two nonmutually exclusive mechanisms that involve a direct antiadhesive effect on polymorphonuclear leukocytes and a negative regulation of ICAM-1 and syndecan-1 expression.     

Antigen recognition in delayed-type hypersensitivity.

It is still uncertain if cell-mediated immune reactions are more or less specific than antibody-mediated reactions. Accordingly, hapten and carrier specificity were examined in delayed hypersensitivity in guinea pigs. Hapten specificity was demonstrated with 2,4-dinitrophenyl (DNP)-guinea pig albumin (GPA), 2,6-DNP-GPA, 2,4,6-trinitrophenyl (TNP)-GPA, and dansyl (DNS)-GPA. Guinea pigs immunized with each of these conjugates were tested 7 days later with the immunogen and the other conjugates. Strong delayed skin responses were highly specific for the immunogen; there were some weak cross-reactions among the nitrophenyl conjugates, no crossre-actions between the DNS and nitrophenyl conjugates, and no responses to unconjugated GPA. Conjugates carrying different numbers (1–45) of 2,4-DNP groups per molecule were all able to elicit specific responses to 2,4-DNP.Carrier specificity in delayed hypersensitivity was confirmed by immunizing with 2,4-DNP-GPA, and challenging with the immunogen, with 2,4-DNP coupled to bovine albumin (BSA), rabbit IgG, ovalbumin, and hemocyanin. Strong responses were seen to the immunogen, a weak response to 2,4-DNP-BSA, and no response to the other conjugates. Specific immune recognition of both hapten and carrier determinants is therefore required for expression of delayed hypersensitivity. These cell-mediated reactions thus appear to be more specific than those of antibody-mediated reactions in solution.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity. II. Suppression of cutaneous delayed-type hypersensitivity by macrophage disappearance reaction

Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 micrograms of EA was injected into these animals and the animals were divided into two groups. One group of animals was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine gamma-globulin (DNP-BGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGG-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supernatants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity: I. Suppression of cutaneous delayed-type hypersensitivity by migration inhibitory factor

The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent column by using anti-guinea pig MIF antiserum. Suppression of cutaneous delayedtype hypersensitivity was achieved by intraperitoneal injection of the MIF fraction into the animals bearing macrophage-rich peritoneal exudates. Skin reactions induced by phytohemagglutinin (PHA) were also suppressed in these animals. Reactivity to skin reactive factor (SRF) was suppressed in these animals as well. The sera obtained from these animals exhibited the inhibitory activity against production of lymphokines from sensitized lymphocytes.     

B-cell-mediated regulation of delayed-type hypersensitivity

We obtained immune sera from mice which received suppressor B cells induced in vitro, injected them into immunized mice, and measured suppression of the delayed-type hypersensitivity (DTH) of these recipient mice. In the recipients, effector-phase suppressor T (Ts) cells were induced, and the action of these Ts cells was antigen-nonspecific. The suppressive material of the sera was adsorbed on a Sepharose column coated with anti-mouse immunoglobulin antibody and acid elution of the column yielded the elute fraction that showed significant suppressive activity. The suppressive activity of the sera was also adsorbed by an antigen-coated Sepharose column, and the eluate from the column had suppressive activity. Moreover, we established antigen-specific monoclonal antibodies, some of which suppressed the DTH in an H-2-nonrestricted way. The isotype or specificity of the antibodies was not related to the suppression, because suppressive and nonsuppressive antibodies belonged to the same immunoglobulin isotype and because the antibodies that recognized the same epitope had different suppressive activities. The Fc portion was not the functional site, because the F(ab')2 fragment had the activity. The suppressive antibody induced effector-phase Ts cells, which had the anti-idiotypic receptor. These findings suggested that antigen-specific antibodies in the immune sera mediated the suppression of DTH by the induction of effector-phase Ts cells in vivo and the idiotype of the antibody stimulated the anti-idiotypic receptor of these Ts cells.     

Regulatory mechanism of delayed-type hypersensitivity in mice. I. Properties of memory cpells and suppressor cells for delayed-type hypersensitivity against ovalbumin.

Delayed-type hypersensitivity (DTH) response in mice induced by sc injection of alum-absorbed ovalbumin (OA) was accelerated and enhanced by priming sc with a low dose of urea-denatured ovalbumin (UD-OA), 2 or more days earlier, whereas it was suppressed by priming sc with a high dose of UD-OA, 0 or more days earlier. The ability in primed mice to accelerate or suppress the DTH response could be transferred antigen specifically into cyclophosphamide (CY)-pretreated recipients or normal recipients by spleen cells from primed mice, but not by the T-cell-depleted spleen cells. Furthermore, the ability of spleen cells to transfer the acceleration or the suppression appeared transiently around 7 or 4 days after priming, although the acceleration or the suppression in donor mice persisted for a much longer time. Pretreatment with CY abolished the suppression of DTH response in high dose-primed mice and resulted in the acceleration of DTH response. These results suggest that the activity of DTH-related memory T cells which accelerate and enhance the response can be inhibited by suppressor T cells for the DTH response.     

Regulatory mechanism of delayed-type hypersensitivity in mice. II. Effect of suppressor cells on the development of memory cells for delayed-type hypersensitivity

Murine lymph node cells (LNC), which we showed previously to noncompetitively inhibit antibody-dependent cellular cytotoxicity (ADCC) to an erythrocyte target, were tested for their ability to inhibit ADCC to a tumor target, EL-4. Both a 4-hr ⁵¹Cr-release cytotoxicity assay and an overnight ¹²⁵IUdR (iododeoxyuridine) postlabeling cytostasis assay were used. Normal autologous lymph node cells inhibited spleen cell-mediated ADCC in both assays. Inhibition by LNC was dose dependent, but comparable numbers of sheep erythrocytes did not inhibit, indicating that LNC-mediated inhibition was not simply a matter of crowding. Inhibitory activity was enriched in LNC after removal of Fc receptor-bearing cells on EA monolayers.     

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.     

Depression of delayed-type hypersensitivity by Corynebacterium parvum: mandatory role of the spleen

Mice pretreated with iv Corynebacterium parvum showed markedly reduced DTH reactivity to subsequently injected SRBC without concomitantly increased antibody levels. No DTH depression occurred if C. parvum was given at the same time, or after, antigen. Neither antigen sensitization at the draining lymph node level nor the subsequent loss of sensitized cells from the node was impaired by iv C. parvum pretreatment. Splenectomy before C. parvum completely abolished its depressive effect, and lymph nodes, even when directly stimulated by local injection of C. parvum, were unable to substitute for the spleen in DTH depression. Increased uptake of sensitized cells by the C. parvum stimulated spleen has been demonstrated, and the discrepancy between the spleen and lymph node performance is discussed in terms of the depression of T-cell activity by C. parvum-activated macrophages described previously.     

Interactions between heterochronic cells in the formation of delayed-type hypersensitivity.

The effect of ageing on lymphocyte and macrophage functional activity in the induction of tuberculin sensitivity was studied in experiments on 440 CBA mice of three age groups. The capacity of old animals to generate delayed-type hypersensitivity following administration of BCG cells was found to be diminished and their lymphocytes caused decreased hypersensitivity on transplantation. The capacity of recipients to reproduce delayed-type hypersensitivity after thymocyte and bone marrow cell transplantation was influenced by the age of transplanted thymocytes. This capacity was markedly suppressed on the recipients of old cells. The antigen presentation function of macrophage did not after significantly as a function of age.     

Ultraviolet radiation-induced immunosuppression of delayed-type hypersensitivity in mice

Ultraviolet (UV) radiation present in sunlight plays a critical role in the initiation and promotion of nonmelanoma skin carcinogenesis and immune suppression. The immune suppressive effects of UV have been identified as a risk factor for skin cancer induction. For these reasons, scientists have focused on elucidating the mechanisms of UV-induced immune suppression to better understand the pathogenesis of skin cancer induction. A hallmark of UV-induced immune suppression is the generation of antigen-specific suppressor T cells. These suppressor cells have been shown to suppress antitumor immunity as well as other cell-mediated responses such as delayed-type hypersensitivity (DTH) reactions. Due to the excessive cost and time involved in traditional UV carcinogenic experiments, scientists have opted to use UV-induced suppression of DTH reactions as a surrogate model. DTH has been, and continues to be, a widely used assay system to measure in vivo immune function. Although somewhat unsophisticated by today's standards, this assay has great advantages because it presents a fast, inexpensive, and reliable model system to help dissect the mechanisms involved in UV-induced immune suppression. Furthermore, the murine model of DTH enables scientists to perform additional procedures, such as adoptive transfer studies with suppressor T cells, which are currently unavailable with human subjects.     

Desensitization of delayed-type hypersensitivity by antigen and specific antibody.

The role of antibody in the desensitization of delayed-type hypersnsitivity (DTH) to dinitrophenylated bovine gammaglobulin (DNP-BGG) was studied in rats. Rats sensitized by a subcutaneous injection of DNP₃₂-BGG in Freund's complete adjuvant (FCA) were desensitized 14 days later with various doses of DNP₃₂-BGG injected intravenously. It was found that only certain doses (100–500 μg) of DNP-BGG effectively desensitized, antigen doses outside this optimum range being ineffective in suppressing DTH. In adoptive cell transfer experiments, it was shown that sensitized peritoneal cells incubated with optimum doses of the antigen in the presence of specific antiserum in vitro failed to transfer the delayed response to normal recipients, whereas the treatment of the sensitized cells with the antigen or with the antiserum separately did not impair the ability of these cells to transfer DTH. The effect of desensitization is specific and is not permanent. The DTH reappears 3–4 wk after desensitizing injection.     

Macrophage-lymphocyte interaction in the formation of delayed-type hypersensitivity

Macrophage-lymphocyte interaction was studied on 121 CBA mice during a 2-hour contact of lymph-node cells of non-immune mice with a monolayer of peritoneal macrophages of BCG-immunized mice and subsequent intravenous administration of 4.10(7) pre-incubated lymphocytes to syngenic recipients. Sensitivity to tuberculin was demonstrated in the recipients by means of blast-transformation reaction of spleen cells (stimulation index was evaluated according to incorporation of 3H-thymidine--SI = 1.32 +/- 0.40) using administration of tuberculin into the paws (Mantoux reaction--MR = 0.11 +/- 0.02 mm) and the cytotoxic effect (CTE) of the lymphocytes on tuberculin-loaded sheep-cell erythrocytes whose disintegration was assessed according to discharge of iron from the target cells (CTE = 13.98 +/- 2.73%). At transfer of intact lymphocytes after contact with non-immune macrophages, SI = 1.046 +/- 0.019; MR = 0.014 +/- 0.002 mm; CTE = 0.214 +/- 0.048%. The treatment of lymphocytes with indomethacin during the contact with macrophages induced idvere changes in the indices of delayed-type hypersensitivity (DTHS). The conclusion has been drawn that the antigen-presenting ability of macrophages can materialize in vitro.     

Regulation of delayed-type hypersensitivity reactions by cyclophosphamide-sensitive T cells.

The onset, intensity, and duration of DTH reactions elicited in mice immunized with either SRBC or products of the major histocompatibility complex can be altered significantly by pretreatment with CY 1 to 2 days before immunization. Such drug pretreatment tends to augment low DTH responses caused by the use of too much antigen and to diminish many responses that are optimal. Thus, pretreatment with CY does not specifically eliminate suppressor cells. Our results are most consistent with the notion that the cellular targets of low doses of CY are positive and negative feedback regulatory cells, which may consist of one population with two effects or, more likely, two distinct cell populations.     

The subset of T lymphocytes mediating delayed-type hypersensitivity in the rat

Populations enriched in each of the major rat T-cell subsets recognized by mouse monoclonal antibodies were prepared from thoracic-duct lymphocytes by affinity chromatography. Such enriched populations obtained from donor rats immunized with BCG were tested for their capacity to mediate delayed-type hypersensitivity (DTH) against purified protein derivative by adoptive transfer to recipient rats. The mediator activity was found in the populations enriched in T cells recognized by the monoclonal antibody W3/25. Thus, the results formally demonstrate that mediating DTH is another function of W3/25-positive T lymphocytes. In addition, the studies show that affinity chromatography is an effective method for preparing bulk quantities of functional lymphocyte populations enriched in the major rat T-cell subsets.