Delayed-type hypersensitivity in mast cell-deficient mice: dependence on platelets for expression of contact sensitivity.

Previous studies of cutaneous T cell-mediated responses in mice have obtained pharmacologic, morphologic, and immunologic evidence pointing to a critical role for local mast cells in release of the vasoactive amine serotonin (5-HT) to mediate early, initiating events that are required for elicitation of these responses. However, the role of mast cells in initiating these T cell-mediated cutaneous responses has been questioned due to the presence of relatively intact delayed-type hypersensitivity responses, such as contact sensitivity (CS), in mast cell-deficient mice whose skin contains only 1 % normal mast cell numbers. The contribution of other potential local sources of 5-HT, such as circulating platelets, at the site of a delayed-type hypersensitivity or CS response in these mast cell-deficient strains, has not been investigated. Therefore, we studied the effect of systemic platelet depletion, produced with an anti-platelet Ab, on blood and tissue levels of 5-HT, and on in vivo T cell-mediated cutaneous sensitivity responses, in W/Wv and Sl/Sld mast cell-deficient mice. The results showed that: 1) platelet depletion severely reduced whole blood 5-HT; 2) tissue levels of 5-HT, in mast cell-deficient mice, depended in large part on the presence of circulating platelets, and 3) specific depletion of platelets markedly suppressed CS responses in both W/Wv and Sl/Sld mast cell-deficient mice, and only moderately reduced CS in normal +/+ congenic mast cell-sufficient controls, but did not decrease CS in beige mice, with platelet granules that are defective in storage of 5-HT. We concluded that platelets may provide 5-HT crucial for the initiation of cutaneous T cell-mediated immune responses, such as CS.

Differential effects of plasma from normal and ectopic gestation on lymphocyte proliferation.

This study investigated immunoregulatory differences as a result of anatomical changes in the fetomaternal interface. Thirty-one female subjects were recruited from a military tertiary-care facility. Ten subjects were non-pregnant while 21 were pregnant (nine normal and 12 ectopic gestations). Immune tests included one-way mixed lymphocyte cultures with and without maternal plasma, phytohemagglutinin stimulation and lymphocyte phenotypes. Basic statistics and a one-way ANOVA using Tuckey's HSD model were employed. Autologous plasma significantly enhanced the allogeneic responses of first-trimester pregnant females with normal gestations to spouses as well as to control male lymphocytes. This enhancement was not observed in either ectopically-pregnant or non-pregnant subjects. Mitogen stimulation and lymphocyte populations were similar in all three groups. The first trimester of normal pregnancy is characterized by the presence of soluble intrauterine pregnancy-specific lymphocyte-enhancing factors. Ectopic gestation lacks this phenomenon.

Borrelia burgdorferi in eastern Virginia: comparison between a coastal and inland locality.

In Virginia, Borrelia burgdorferi was more prevalent in a site along the Atlantic Ocean, near Maryland, than in an inland site near Williamsburg and Yorktown. At the coastal site on Assateague Island, B. burgdorferi was isolated from 4.2% of 475 animals sampled, including four species of small mammals. Serologic tests indicated that 25-37% of the small rodents assayed had been exposed to B. burgdorferi. Immunofluorescence antibody assays specific for B. burgdorferi showed spirochete infection in Ixodes scapularis and Dermacentor variabilis but not in other species of ticks also examined from this site. At another coastal site (Parramore Island), no evidence of Peromyscus leucopus was found, no immature specimens of I. scapularis were collected, and no isolations were made from numerous raccoons or small mammals sampled. Borrelia burgdorferi infection was found in one I. cookei nymph, but not in numerous specimens of I. scapularis or other tick species from this locality. At the inland site between Williamsburg and Yorktown, B. burgdorferi was isolated from two small mammal species and antibodies to B. burgdorferi were found in only 7-10% of the small mammals sampled. Ixodes scapularis were less abundant at this locality than at the Assateague Island site. Borrelia burgdorferi spirochetes were found in I. scapularis and a single nymph of Amblyomma americanum, but not in any of numerous specimens of four other species. Infection with B. burgdorferi was found in 20% of unfed adult I. scapularis from vegetation, but in only 0.2% of numerous adults from hunter-killed deer. Infection in immature ticks was much lower than at Assateague Island. Borrelia burgdorferi may be more prevalent along the Atlantic coast than in inland areas. Isolations, seroprevalence, immature I. scapularis densities, and spirochete infection rates in ticks were higher at the Assateague Island site than the Williamsburg/Yorktown site. Consequently, the risk of human exposure to Lyme disease may be higher in some parts of the coastal area than elsewhere in Virginia. Overall, B. burgdorferi is less intense in Virginia than in the northeastern United States.

A role for platelet release of serotonin in the initiation of contact sensitivity.

Finding contact sensitivity (CS) responses that were fairly normal in ear swelling, and in serotonin (5-HT) dependence in mast-cell-deficient mice, led to experiments to determine whether platelets supplemented mast cells as a source of 5-HT in CS. Severe depletion of platelets, and consequently blood 5-HT, with antiplatelet antibody, strongly inhibited CS, especially in mast-cell-deficient mice, suggesting that platelets supplemented mast cells. Furthermore, human platelets sensitized in vitro with anti-(tri-nitro-phenyl) IgE, and transferred intravenously together with isolated late-acting effector T cells, provided CS initiation due to local 5-HT release. Similar, IgE-dependent in vitro release of 5-HT was C dependent. These findings establish the importance of antigen-specific platelet release of 5-HT in CS initiation.

Involvement of an axonal reflex in IgE-mediated inflammation in mouse skin.

A neurogenic component of IgE-mediated inflammation was demonstrated in mice by footpad denervation. Footpad swelling was reduced 26% following sciatic nerve transection, but unaffected by rhizotomy or spinal nerve transection. These data provide in vivo evidence that an axonal reflex is involved in IgE-mediated inflammation and completed distal to the cell bodies of the sensory neurons located in the lumbar spinal ganglia. Furthermore, depletion of neuropeptides with capsaicin also reduced IgE-mediated swelling by 26%, indicating that unmyelinated axons are involved in the neurogenic component of IgE-mediated inflammation.

Evidence of a neurogenic component during IgE-mediated inflammation in mouse skin.

IgE-mediated inflammation was measured in mouse footpads that lacked sciatic innervation. Mice were passively sensitized with a monoclonal antibody, IgE anti-dinitrophenol, or were immunized for specific IgE production. Antigen-induced swelling in the denervated footpads was reduced 23-39% when compared to sham or untreated controls. Reduced IgE-mediated swelling responses were attributed to the loss of a mast cell-nerve interaction and not to blood vessel sensitivity to vasoamines. Furthermore, electrical stimulation of the distal segment of the sciatic nerve completely restored IgE-mediated inflammation. These data provide in vivo evidence that peripheral nerves participate in cutaneous IgE-mediated swelling reactions with the net effect of increasing inflammation.

Murine immune responses and immunization against Polyplax serrata (Anoplura: Polyplacidae).

Mice with restricted grooming capabilities were infested with the solenophagous louse, Polyplax serrata (Burmeister). Louse burdens on Cox/Swiss and C3H/HeSN mice increased for approximately 1 mo, reaching burden/host weight ratios of 1.14 and 1.26 mg/g, respectively, followed by a steady decline. Fifty days after initial ectoparasite contact, both strains were resistant to lice. Resistance was anamnestic, lasting several months with second infestation weights reduced by 98 and 78% on Cox/Swiss and C3H/HeSN, respectively. Furthermore, mice were systemically resistant because infestations on naive body sites of resistant hosts were reduced by 59%. Host resistance was associated with the development of antilouse immune responses. After the first week of a primary infestation, the draining lymph nodes contained cells that proliferated in vitro to louse antigens. Skin responses to louse antigens were also detected: (1) delayed, (2) immediate and delayed, and (3) no significant reactivity on days 19, 34, and 54, respectively. The presence of systemic antilouse responses provided an immunologic basis for immunization against lice. Intradermal injections of soluble louse components reduced primary infestation weights by 62%. Immunized mice had immediate and delayed skin responses containing an inflammatory infiltrate 1 wk following immunization. This study, using the natural host of P. serrata, demonstrates an inducible, anamnestic immune component in louse resistance.

Immune serum from mice contact-sensitized with picryl chloride contains an antigen-specific T cell factor that transfers immediate cutaneous reactivity.

This report describes an activity in serum from mice that were contact-sensitized with picryl chloride (PCl) 1 to 4 days earlier. Immune serum, when given i.v., transfers the ability to elicit an immediate hypersensitivity-like ear swelling reaction in naive recipients following local challenge with PCl. This serum activity is due to an antigen-binding T cell factor that shares some properties with IgE antibody. The activity is antigen specific, and due to an antigen-binding moiety that is heat labile (56 degrees C, 4 h). However, unlike IgE antibody the serum activity is resistant to reduction and alkylation, and is retained by columns of Sepharose beads coupled with polyclonal or monoclonal antibodies that react with antigen-specific T cell factors from other systems. These columns did not retain IgE antibody activity in our experiments. Importantly, the serum activity was not retained by columns linked with antibodies directed to mouse immunoglobulins, which do retain IgE activity. We conclude from these data that the activity in PCl immune serum is not caused by IgE antibody, and is due to the presence of the previously described antigen-specific T cell factor (PCl-factor), that can activate serotonin-containing cells, such as mast cells, to release the vasoactive amine serotonin. PCl-factor transfers the ability to elicit an immediate hypersensitivity-like reaction that is an early component of delayed-type hypersensitivity. The presence of this T cell factor in the serum of actively sensitized mice provides a means to sensitize tissues throughout the body for this required, initial, serotonin-dependent component of delayed-type hypersensitivity reactions.

Interaction of antigen-specific T cell factors with unique "receptors" on the surface of mast cells: demonstration in vitro by an indirect rosetting technique.

Picryl (trinitrophenyl) chloride (PCL) contact sensitization of mice induces T cells that release an antigen-binding T cell factor (PCLF) that plays an important role in the initiation of contact sensitivity responses, in part via activation of mast cells. The current study employs an in vitro indirect rosette assay to demonstrate that PCLF can interact with the mast cell surface. Sheep red blood cells (SRBC) were hapten conjugated with trinitrophenyl (TNP), dinitrophenyl (DNP), or oxazolone (OX). When TNP-conjugated SRBC were coated with PCLF, monoclonal anti-DNP IgE, or anti-DNP IgG1, they produced 40 to 50% rosettes with purified normal mouse peritoneal mast cells. Analogous antigen-binding factors, from lymphoid cells of OX and dinitrofluorobenzene contact-sensitized mice, gave similar mast cell rosetting levels with OX-SRBC and DNP-SRBC, respectively. PCLF demonstrated a high degree of hapten specificity in that it formed rosettes with TNP-SRBC but not with DNP-SRBC, unlike IgE and IgG1, or DNPF, which formed rosettes with either SRBC type. Similarly, soluble TNP-BSA could inhibit PCLF rosette-forming capacity, but soluble DNP-BSA could not. In addition to mouse mast cells, PCLF formed rosettes with rat basophil leukemia cells, mouse peritoneal exudate macrophages, mouse alveolar macrophages, and J 774 cultured mouse macrophages; it did not form rosettes with rat mast cells, rat alveolar macrophages, or mouse spleen cells. Thus, PCLF-formed rosettes were antigen specific, relatively species specific, and mast cell/macrophage specific. PCLF-mediated rosette-forming activity could be detected in the presence of nanogram quantities of PCLF. More than 10 times greater IgE was needed to produce IgE-mediated rosettes. Reduction and alkylation eliminated the rosetting activity of IgE, but the rosetting activity of PCLF was not affected. PCLF, but not IgE rosette-forming activity, could be removed by and eluted from affinity columns linked with a monoclonal antibody specific for T cell-derived antigen-binding factors, whereas PCLF rosetting activity was not retained by an anti-immunoglobulin affinity column. Preincubation of mast cells with rat myeloma IgE or mouse monoclonal IgE of various specificities blocked IgE rosettes but not PCLF-induced rosettes. Other immunoglobulin isotypes likewise did not block PCLF rosettes. However, PCLF rosettes could be blocked by preincubation of mast cells with OX factor (OXF),and OXF-mediated rosettes could be blocked similarly by PCLF. These results suggest that the antigen-binding T cell factor PCLF interacts with a unique receptor on the surface of mouse mast cells.

Use of micrometers and calipers to measure various components of delayed-type hypersensitivity ear swelling reactions in mice.

The choice of the type of instrument to measure delayed-type hypersensitivity (DTH) in mice, as assayed by ear swelling reactions, influences the experimental results. When a caliper that applies little pressure to the ears is employed, DTH reactions in ears of mice sensitized to picryl chloride show an early onset at 2 h after challenge, comparable swelling at 4 h and a slow rise to a 24 h classical peak response thereafter. In contrast, 3 different micrometers that apply more pressure to the ears reveal a biphasic pattern of ear swelling reactions in mice immunized and challenged with picryl chloride. The early component of DTH measured by these micrometers peaks 2 h after challenge. Thereafter the measured ear thickness declines, and the onset of the classical delayed reaction is detected at 12 h after ear challenge. Yet another instrument, that in contrast to the caliper and micrometers mentioned above, applies all the pressure to only a very restricted area of the ear, fails to detect an early swelling reaction; the delayed reaction is first detected at 12 h after ear challenge and rises thereafter to a 24 h peak. The differences in outcome of the assays using the different instruments indicate that the early component or DTH reactions differs from the late component of DTH reactions in that the early swelling is easier to compress when pressure is applied by the instrument used for measurement. This is probably caused by the fact that the late reactions are due to a cellular infiltrate, whereas the early reactions are edematous in character, and are due to accumulation of plasma components.