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To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3Ralpha (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) >/= eotaxin-2 = eotaxin-3 >/= eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1alpha > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.

The relationship of expression of the C-C chemokines eotaxin, eotaxin 2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4 to the kinetics of infiltrating eosinophils, basophils, and other inflammatory cells was examined in allergen-induced, late-phase allergic reactions in the skin of human atopic subjects. EG2+ eosinophils peaked at 6 h and correlated significantly with eotaxin mRNA and protein, whereas declining eosinophils at 24 h correlated significantly with eotaxin-2 and MCP-4 mRNA. In contrast, no significant correlations were observed between BB1+ basophil infiltrates, which peaked at 24 h, and expression of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 or elastase+ neutrophils (6-h peak), CD3+ and CD4+ T cells (24 h), and CD68+ macrophages (72 h). Furthermore, 83% of eosinophils, 40% of basophils, and 1% of CD3+ cells expressed the eotaxin receptor CCR3, while eotaxin protein was expressed by 43% of macrophages, 81% of endothelial cells, and 6% of T cells (6%). These data suggest that 1) eotaxin has a role in the early 6-h recruitment of eosinophils, while eotaxin-2 and MCP-4 appear to be involved in later 24-h infiltration of these CCR3+ cells; 2) different mechanisms may guide the early vs late eosinophilia; and 3) other chemokines and receptors may be involved in basophil accumulation of allergic tissue reactions in human skin.

Hypersensitivity reactions containing significant infiltrates of basophils occur in a large proportion of allergic diseases such as contact dermatitis, atopic dermatitis, and allergic rhinitis, and are quite deleterious. However, the potential usefulness of such responses can be recognized in similar reactions in guinea pigs responding to tissue invasion by complex multicellular parasites in which interactions occur between thymic-derived T lymphocytes, antibodies, and basophils in immune resistance responses. Perhaps inappropriate and deleterious allergic responses to pollens, chemicals, and insects is the price that we must pay for the ability to reject complex parasites.

We have described functional and biochemical characteristics of a distinct T-cell dependent guinea-pig basophil-stimulating factor (BSF), measured using a sensitive 7-day bone marrow culture assay, standardized with high-activity BSF present in serum-free splenic cell-conditioned medium (CM). In the present studies, the in vivo relevance of BSF was explored during protocols of induction of peripheral blood or tissue basophil responses to ovalbumin (OA) injection or Amblyomma americanum tick feeding. Pooled immune serum, taken from OA-injected inbred or outbred animals during induction of blood and marrow basophilia, contained an in vitro inhibitor to BSF at high concentrations and BSF-like activity at low concentrations; maximal stimulation of histamine synthesis by bone marrow cells in vitro was found in the presence of Day 4 OA-immune serum. In vivo studies in the OA model demonstrated maximal serum BSF-like activity at 48-72 hr before peak bone marrow basophil response, followed by a levelling off to 50% of maximum at 2 weeks. In the tick model, serum BSF-like activity was present in Day 8, but not Day 1, post-primary infection and was maximal at Day 3 post-secondary infection; post-primary Day 1 serum was inhibitory to basophil growth in vitro. These observations suggest that BSF regulates the appearance of basophils in response to antigen in vivo by an effect on basophil progenitors. The observations stress the potential application of guinea-pig models to understanding the regulation of basophil production in allergic disorders.

Cutaneous basophil hypersensitivity (CBH) was studied in guinea pigs by using simplified histologic techniques. Animals immunized with oxazolone or picryl conjugates of keyhole limpet hemocyanin (KLH) emulsified with complete (CFA) or incomplete Freund's adjunvant (IFA) were found to have hapten-specific cutaneous basophil reactions when skin tested at 1 week with oxazolone or picryl chloride contant painting or intradermal injection of oxazolone or picryl-conjugated human serum albumin, respectively. Thus, hapten-specific cutaneous basophil reactions were present in guinea pigs immunized with CFA for classical delayed hypersensitivity, and in animals immunized with IFA for Jones-Mote reactions. Hapten-specific 24-hr cutaneous basophil reactions were passively transferred with immune serum from donors sensitized with conjugates of oxazolone or picryl-KLH in CFA or IFA, and with serum from oxazolone contact-sensitized animals as well. As little as 0.5 ml sera obtained from donors 1 week after immunization could systemically transfer cutaneous basophil reactions. It is likely that hapten-specific cutaneous basophil reactions are mediated by small quantities of serum antibodies. We conclude that antibody-mediated cutaneous basophil reactions may be distinctive hypersensitivity responses that can be distinguished from classical anaphylactic, Arthus, and delayed hypersensitivities. It is suggested that CBH reactions are heterogeneous and that antibody products of B lymphocytes, and factors probably derived from T lymphocytes, play a role in basophil accumulations at cutaneous hypersensitivity reactions.

We have induced a basophil hypersensitivity reaction in the upper tarsal conjunctiva of the guinea pig by methods that induce a comparable basophil hypersensitivity reaction in the flank. The inflammatory cell infiltrate in this reaction contained large numbers of basophils and eosinophils with accompanying neutrophils and monocytes. Ocular tissue can serve as a priming site for systemic immunization and also for elicitation of a secondary flare after challenge with antigen. Very few inflammatory cells were observed in the cutaneous epithelium of either primary or secondary flares. In contrast, the mucosal stroma and epithelium contained large numbers of inflammatory cells (basophils, eosinophils, and neutrophils), suggesting directed cellular movement onto the ocular surface. The lesion of ocular basophil hypersensitivity in the guinea pig has features in common with two human eye diseases, vernal conjunctivitis and contact lens-associated giant papillary conjunctivitis. We hypothesize that the acute basophil hypersensitivity reactions of the conjunctiva are transformed into chronic inflammatory and proliferative states in vernal conjunctivitis and giant papillary conjunctivitis.

Many delayed-type reactions contain large infiltrates of basophils whose function is unknown. We have studied these cutaneous basophil hypersensitivity (CBH) reactions in guinea-pigs to ascertain whether basophils that are recruited to delayed reaction sites could be triggered for immediate reactivity. We compared 24 h CBH reactions with nearby skin for immediate hypersensitivity by challenging each site with small amounts of antigen. CBH sites had augmented immediate increases in vascular permeability detected by extravasation of Evan's blue dye. The ability to elicit this augmented anaphylactic phenomenon correlated with the local presence of basophils, and light microscopy at CBH reactions 15 min after antigen challenge showed a 50% decline in basophil counts. Electron microscopy showed that progressive anaphylactic-type degranulation of local basophils occurred within minutes following reintroduction of antigen. There was fusion of vacuoles containing granules, exocytosis of granules, and dissolution of granules, without ultrastructural disruption of cellular integrity. These results establish that basophils in CBH reactions can be triggered with soluble antigen to undergo anaphylactic degranulation, with the immediate release of vasoactive mediators. We have termed this phenomenon 'cutaneous basophil anaphylaxis'. Thus, one function of basophils at sites of delayed hypersensitivity may be to provide the potential for augmented, local, immediate anaphylactic reactivity.

In guinea pigs, macroscopic cutaneous reactions to initial (primary) Ixodes holocyclus feeding were first apparent at 96 hr post-tick attachment, peaked at 7 days (5 mm), and were gone by Day 14. Microscopic analyses of these primary tick feeding sites at 12, 24, 48, 72, and 96 hr post-attachment revealed the dominance mononuclear cells (63-94% of the infiltrate) at all times. Neutrophil levels were high initially (34% of the infiltrate), but quickly subsided to 6-15% of the cellular response. Eosinophils were essentially absent from primary sites, comprising only 1-3% of the infiltrate. Basophils were absent initially, but accumulated in small but significant numbers (12% of the infiltrate) at the epidermal-dermal border by 96 hr post-tick attachment, 3 days prior to maximum erythematous skin reactions. In sensitized and challenged (secondary) animals, erythematous reactions in response to secondary tick feeding were apparent as early as 18 hr post-tick attachment with peak responses at 48 hr (3-4 mm). Cutaneous leukocyte responses to challenge feedings were quantitated at 12, 24, 48, and 72 hr post-tick attachment and consisted initially (12-24 hr) of a strong mononuclear cell response (72-79% of the infiltrate) that was replaced by a dominant basophil response at 48 and 72 hr. Strong cutaneous basophil responses coincided with a significant level of tick rejection. Eosinophils and neutrophils were virtually absent from these secondary responses.(ABSTRACT TRUNCATED AT 250 WORDS)

When antigen is injected into a 24-hr cutaneous basophil hypersensitivity (CBH) reaction of an actively sensitized guinea pig, local basophils degranulate and release histamine. This reaction is called cutaneous basophil anaphylaxis and may be antibody mediated. We now report passive sensitization of basophils at CBH sites by systemic transfer of anti-picryl immune serum. Keyhole limpet hemocyanin- (KLH) immunized animals were skin tested with KLH to elicit 24-hr CBH reactions at day 7. Anti-picryl serum was injected i.v. at various times. On day 7, blue dye was injected i.v., and then 24-hr CBH sites vs nearby normal skin were challenged with 0.1 microgram picryl-human serum albumin (Pic-HSA). An immediate increase in vascular permeability (blueing) was noted at normal skin sites due to systemic passive cutaneous anaphylaxis (PCA), and augmented blueing occurred at CBH sites compared with normal skin. Systemic passive sensitization of CBH sites occurred when antiserum was administered as little as 1 hr before challenge of CBH site. However, local administration of anti-picryl serum (as in a local PCA reaction) was not able to sensitize tissue basophils, whether antigen was administered locally or systemically. The serum factor that mediated cutaneous basophil anaphylaxis was heat-stable (56 degrees C X 4 hr) 7S IgG1 antibody. Electron microscopy of Pic-HSA-challenged CBH sites in animals that received IgG1 antibody showed that local basophils undergo anaphylactic degranulation by exocytosis. These studies suggest that basophils arriving at CBH reactions are sensitized for anaphylactic function by antibody that can be acquired in the circulation, but possibly not at the local site.

Previous studies have demonstrated a failure to elicit delayed hypersensitivity skin responses in neonatal guinea pigs despite apparent immunologic competence of their lymphocytes and monocytes. The ability of neonatal guinea pigs to manifest cutaneous basophil hypersensitivity (CBH) responses was examined. Neonates were competent to express macroscopic and microscopic aspects of these delayed reactions. Strong cutaneous basophil responses were elicited in newborns (0 to 2 days old) in a hapten-specific manner after intravenous transfer of small amounts of immune serum (0.5 ml) obtained from adult donors. Also, neonatal guinea pigs actively immunized at birth with hapten-carrier conjugates emulsified with incomplete Freund's adjuvant (IFA) and skin tested when 1 week old had 24-hr hapten-specific cutaneous basophil reactions and CBH reactions to the carrier protein as well. Compared with adult CBH reactions, neonatal responses had equal concentrations of basophils, fewer monocuclear cells, less macroscopic erythema, and almost no induration. Hapten-specific CBH reactions also contained significant infiltrates of eosinophils which were more prominent in the ear skin vs flank skin and in neonates vs adults. Immunization with complete Freund's adjuvant (CFA) resulted in a marked difference between adults and neonates; adult PPD reactions were quite indurated and contained many more monoculear cells and few basophils, whereas neonatal PPD reactions were flat, erythematous, nonindurated, and contained relatively few mononuclear cells and more basophils. Thus, neonatal tuberculin reactions elicited by PPD in animals immunized with CFA were examples of CBH. Skin testing nonimmune guinea pigs with phytohemagglutinin (PHA) also revealed marked differences in 24-hr cutaneous reactions between adults and newborns. Adults had indurated and erythematous reactions which contained approximately 20% basophils and 80% mononuclear cells, whereas similar PHA skin tests in newborns elicited small macroscopic reactions, which microscopically showed large infiltrates containing approximately 80% basophils and 20% mononuclear cells. It was concluded that neonatal guinea pigs were not only competent to manifest basophil-containig delayed-type reactions, but that cutaneous basophil responses were preferentially elicited in these animals under a variety of circumstances. These results underline the fact that basophil accumulations are one aspect of delayed skin test responses and that the regulation of the arrival of these cells in neonates is different from that in adult guinea pigs.

Previous studies have shown that injection of antigen into 24 hr cutaneous basophil hypersensitivity (CBH) reaction sites results in immediate degranulation of local basophils and release of mediators, such as histamine, that increase vascular permeability. In the current study, cell counts were made at these antigen-challenged CBH sites, 4, 8 and 24 hr later, and demonstrated a prolonged reduction of basophil numbers following antigen challenge. These results were interpreted to signify that mediators released by degranulating basophils led to inhibition of subsequent basophil accumulation. Local eosinophil infiltration was not inhibited, and in some cases was augmented. Local injection of histamine into CBH sites did not mimic the effect of antigen. It was concluded that one of the factors regulating accumulation of basophils at CBH sites was degranulation of basophils that had arrived previously.

Cutaneous basophil hypersensitivity (CBH) reactions are heterogeneous delayed time course basophil-rich responses that can be mediated by either T cells, B cells, or serum antibodies. The current study examined the mechanism by which antibodies mediate CBH in guinea pigs. Fc competition experiments were constructed by passively transferring mixtures of anti-KLH serum and normal heterologous gamma-globulins. It was found that rabbit IgG and its isolated and purified Fc fragment [but not the (Fab')2 fragment] inhibited the ability of guinea pig immune serum to transfer CBH. Concurrent inhibition of transferred KLH-specific CBH and systemic passive cutaneous anaphylaxis (PCA) reactions by rabbit IgG or its Fc fragment, and not by sheep or bovine gamma-globulins, indicated that Fc receptors on cutaneous mast cells were probably involved in both CBH and PCA. It was also found that the basophil aspect of delayed cutaneous responses elicited by PHA was inhibited by Fc competition maneuvers. This could mean that some forms of apparently T cell-mediated CBH may be T cell dependent, but via secretion of molecules that bind to Fc receptors, as seems required in antibody-mediated CBH.

Jones-Mote reactions are delayed, erythematous, and mildly indurated cutaneous reactions originally described in humans sensitized by skin injection of heterologous proteins. Similar reactions in guinea pigs contain many basophils and are called cutaneous basophil hypersensitivity. In contrast, guinea pigs immunized with mycobacterial adjuvants have classical tuberculin-type delayed hypersensitivity reactions, which contain few basophils. This has led to a new classification of delayed responses, based largely on the presence or absence of basophils. We induced sensitization for Jones-Mote reactions in 20 normal humans by intradermal injections of keyhole limpet hemocyanin. Skin tests with KLH 1 wk later showed erythematous and indurated delyaed reactions in all subjects. Rebuck skin windows showed specific accumulations of basophils with a delayed time-course in 18 of 20 subjects. In 12 normals sensitized with oxazolone-keyhole limpet hemocyanin conjugates, skin reactions and in vitro lymphocyte stimulation showed carrier and not hapten specificity, suggesting that cutaneous responses were probably mediated by T cells. A comparative study of strongly positive PPD skin tests in patients with tuberculosis showed significant basophil accumulations in five of nine subjects. Thus, basophils occurred in human tuberculin and Jones-Mote reactions and were not a distinguishing feature of Jones-Mote reactions. We suggest that the occurrence of basophils at delayed reactions is under complex regulation and that basophil accumulations are an aspect of delayed hypersensitivity, rather than an indication of a distinctive and separate response.

Guinea pigs immunized with protein antigens emulsified with complete Freund's adjuvant (CFA) and skin tested at 3-4 wk have classical tuberculin-type delayed hypersensitivity (DH) reactions with few basophils present. However, recipients of T cells from these animals have delayed responses containing large basophil infiltrates and thus resemble basophil-rich cutaneous basophil hypersensitivity (CBH) responses that are elicited in animals immunized without CFA. This suggests that animals immunized with CFA have T cells with basophil-recruiting capacity but that this activity is suppressed. Using a transfer system, we found that immune serum from donors immunized with CFA had the ability to suppress the basophil-recruiting capacity of immune T cells. When immune serum and peritoneal exudate cells from guinea pigs immunized with CFA were co-transferred intravenously to normal recipients, the cell-mediated transfer of basophil-rich responses was suppressed. The responsible serum factor was antigen nonspecific, had an approximately 70,000 mol wt, and acted preferentially on cells from donors that express basophil-poor DH responses. Thus, tuberculin-type delayed hypersensitivity and CBH might be mediated by a common T cell, but the resulting basophil component of the delayed response depends on the modulation of T cell recruitment of basophils by factors in CFA-immune serum.

Basophils infiltrate the skin and other tissues as part of T lymphocyte and/or antibody-mediated immune responses to certain protein antigens, viruses, tumors, and parasites. Although basophils may comprise a significant fraction of leukocytes in these reactions, their precise role has been poorly understood. Guinea pigs expressing acquired immunity to the tick Amblyomma americanum develop basophil- and eosinophil-rich cutaneous inflammatory responses at tick feeding sites, and tick rejection is associated with extensive local basophil degranulation. We report that a specific anti-basophil serum (ABS) eliminate basophils at tick feeding sites and abolished immunity to A. americanum. ABS does not react with eosinophils and did not reduce blood or bone marrow eosinophils; however, ABS-treated animals had diminished eosinophils at tick feeding sites, perhaps because these sites were deficient in basophil-derived eosinophil-chemotactic factors. AES treatment markedly reduced feeding site eosinophils, did not affect basophil levels, and partially impaired tick resistance. Taken together, our experiments suggest cooperation between basophils and eosinophils in the expression of immunity to ticks in guinea pigs.

Electron microscopy has been used to monitor cellular activity in dermal lesions elicited by larval Rhipicephalus appendiculatus feeding on actively sensitised guinea pigs and recipients of immune serum. The early primary response is characterised by mononuclear cells, many of which appear to be activated fibroblasts. Collagen deposition is enhanced as the reaction progresses. Granulocytes of all types appear in the lesion between 18 and 96 hr but they show no evidence of degranulation. Free, membrane-bounded eosinophil and basophil secretion granules may, however, be identified in the dermis at day 5 or 6, but they seem to be liberated as a consequence of cellular disruption, rather than active degranulation. Some feeding sites resume a normal morphology by day 7. Lesions induced in actively sensitised hosts by a secondary feeding tick population are dominated by basophils. These cells begin to infiltrate the dermis within 6 hr and they show evidence of anaphylactic degranulation at 12 hr. Maximal release of membrane-free secretion granules occurs at about 18 hr post-attachment, at which time eosinophils become prominent. Degranulating basophils show a reduction in numbers from 24 to 96 hr, and phagocytic macrophages ingest residual granules and cellular debris. Guinea pigs sensitised with immune serum and subjected to challenge exhibit lesions similar to but less dramatic than those of actively sensitised and challenged animals. Anaphylactic degranulation of basophils occurs both in the dermis and within blood vessels. The immunological consequences of these events are discussed in relation to other models of cutaneous basophil hypersensitivity.

Previous studies have established that recruitment of basophils to sites of tick feeding in guinea pigs is required to effect immune resistance. In the current study, actively sensitized guinea pigs treated three times daily with H-1 (mepyramine) and H-2 (cimetidine) histamine receptor antagonists, during the challenge tick infestation period, expressed normal resistance to Amblyomma americanum larvae. Similarly, naive guinea pigs treated with anti-histamines four times daily, beginning 7 days before transfer of immune serum and tick challenge and continuing through the tick infestation period, also expressed normal antibody-mediated resistance to A. americanum. These results indicated that histamine was not an important basophil mediator of the resistance response. Ticks allowed to feed on tissue rich in basophils that were induced by sensitization and subsequent local challenge with non-tick protein antigen, keyhole limpet hemocyanin (KLH), expressed normal yield. Ticks that fed on similar tissue rich in basophils induced by sensitization and challenge with KLH, in which the basophils expressed anti-picryl specificity due to systemic passive transfer of anti-picryl antibodies, were rejected when basophils were induced to degranulate by i.v. challenge with picryl antigen at 6 hr (29% rejection), 12 hr (18% rejection), 24 hr (22% rejection), and 48 hr (37% rejection) post-tick attachment. However, basophil degranulation at 18, 72 and 96 hr post-tick attachment had no adverse effect on tick feeding. These hosts were protected from systemic anaphylaxis by treatment with the anti-histamine mepyramine. Release of histamine occurred at tick feeding sites, but vasoactive effects were blocked by mepyramine treatment as evidenced by a lack of increased vascular permeability (bluing) at these sites compared with non-tick-infested tissues, or to cutaneous basophil hypersensitivity (CBH) sites of animals not protected with mepyramine. These results indicate that local recruitment and subsequent degranulation of basophils via immune mechanisms dependent on non-tick antigens can lead to tick rejection, and that basophil-derived mediators other than histamine are involved in this immune resistance response to A. americanum ticks. The identity of the crucial basophil mediator(s) is not known. The significant susceptibility of ticks to basophil-mediator release at 6 to 12 hr and 24 to 48 hr post-attachment coincides with the tick attaching and fast-feeding phases, respectively, suggesting that these phases of tick parasitism are particularly susceptible to the effect of basophil mediators other than histamine.

We studied binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells. We found two types of serotonin binding protein in RBL cells. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 X 10(-6) M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD1 = 4.5 X 10(-8) M; KD2 = 3.9 X 10(-6) M) did not bind to Con A. Moreover, binding of [3H]serotonin to protein of peak I was sensitive to inhibition by reserpine, while binding of [3H]serotonin to protein of peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract peak I than peak II protein. Neither peak I nor peak II protein resembled the serotonin binding protein (SBP) that is found in serotonergic neurons of the brain and gut. Electron microscope radioautographic analysis of the intracellular distribution of [3H]serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules. No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but peak I protein may be associated with the granular membrane while peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules.

Cutaneous basophil hypersensitivity (CBH) reactions are a heterogeneous group of delayed time course basophil-rich immune responses that can be mediated in the guinea pig by T cells, B cells, or IgG1 antibody. This study examined whether guinea pig IgE antibody could also mediate CBH reactions. IgE antibody to picryl or oxazolone determinants was induced by immunizing Hartley strain guinea pigs pretreated with cyclophosphamide. Hyperimmune serum from these animals was passed through a heavy chain-specific anti-IgG1 affinity column. The presence of IgE anti-hapten antibody in the filtrate fraction was verified by passive cutaneous anaphylaxis (PCA) testing with a 7-day period of local passive sensitization and by the heat lability (56 degrees C, 4 hr) of PCA activity. This IgE-rich fraction and the IgG1 fraction eluted from the column with base (0.2 M Na2CO3, pH 11.3) were transferred i.v. to separate groups of normal guinea pigs. Both fractions mediated delayed time course reactions that contained basophils. Macroscopic and microscopic reactions mediated by the IgE-rich fraction were abolished with heat (56 degrees C, 4 hr). Thus, two antigen-specific factors in guinea pig serum can mediate delayed time course basophil-containing reactions: IgG1 and IgE antibodies. IgE-mediated CBH reactions are similar to the late-phase reaction that follows IgE-dependent wheal-and-flare reactions in humans. The finding that guinea pig IgE can mediate a late reaction that contains basophils makes this a possible model for the human late-phase response, and suggests that some forms of CBH may play a role in human allergic disease.

An in vitro histamine release assay was used to test the hypothesis that passive sensitization of circulating basophils is associated with the activity of immune serum that transfer the ability to elicit cutaneous basophil hypersensitivity (CBH) reactions. Systemic i.v. transfer of several types of immune sera that mediate CBH also led to passive sensitization of circulating basophils for antigen-specific release of histamine in vitro. In addition, we found that immune serum passively sensitizes basophils in vitro. Thus immune sera had three activities that are probably interconnected: sera will 1) passively transfer CBH in vivo, 2) passively sensitize basophils in vivo, and 3) passively sensitize basophils in vitro. These results suggest that passive sensitization of circulating basophils by immune serum contributes to the mechanism by which antibodies transfer the ability to elicit CBH reactions.

Guinea pigs acquired resistance to Amblyomma americanum larval ticks after one infestation, resulting in 46% tick rejection when challenged. Intravenous transfer of immune serum from twice-infested hosts to naive animals conferred a significant level of immunity resulting in 18 to 30% tick rejection. The minimum effective dose of serum was 3 ml per recipient, and heating the serum at 56 degrees C for 4 hr had no effect on serum activity. Fractionation of whole immune serum by gel filtration chromatography (Sephadex G-200) and ion -exchange chromatography (DEAE) demonstrated resistance activity to be in the IgG- and IgG1-containing fractions, respectively. Passage of whole immune serum through a heavy chain-specific rabbit anti-guinea pig IgG1 affinity column removed anti-tick activity and decreased the cutaneous basophil response to tick feeding by 70% in recipients. The ability to transfer both resistance to tick feeding and a significant cutaneous basophil response was eluted from the affinity column with 0.2 M Na2CO3, pH 11.5. In addition, immune serum raised against larval Ixodes dammini ticks, but not larval Rhipicephalus sanguineus ticks, was also effective at reducing feeding by larval Amblyomma americanum ticks, indicating that antibodies mediating resistance can be cross-reactive with antigens of different tick species and genera. This study demonstrates that IgG1 antibodies are responsible for the ability of immune serum to transfer cutaneous basophil-associated immune resistance against tick feeding in guinea pigs.

Bone marrow-derived blood basophils are recruited into the tissues by immuno mechanisms in a variety of delayed time-course hypersensitivity responses. In the skin these are called cutaneous basophil hypersensitivity (CBH) reactions. In guinea pigs, it is now established that the elicitation of CBH is dependent on T cell- and/or (antibody)-triggered mechanisms. Both are subject to modulation. T cell-mediated CBH seems to be suppressed in basophil-poor tuberculin-type reactions. B cells mediate CBH via antibody of IgG1 isotype through mechanisms that involve Fc receptors, which can be competitively blocked. After basophils arrive at a CBH reaction they can be triggered by antigen to immediately release mediators such as histamine. Thus, one consequence of the arrival and accumulation of basophils at delayed hypersensitivity reactions is to augment the anaphylactic potential of a given tissue site. In reactions to parasites, release of mediators by tissue basophils seems to aid in the expulsion of these multicellular organisms, In addition, histamine released by recruited basophils, or by locally resident mast cells, may modulate some delayed reactions through stimulation of histamine-2 receptors on cells such as T lymphocytes. In mice, mast cell release of serotonin and subsequent stimulation of the local vasculature seems to be required to allow diapedesis and tissue accumulation of various bone marrow-derived accessory leukocytes in delayed-type hypersensitivity responses. Thus, basophils and mast cells, and their release of mediators such as vasoactive amines, are involved in the onset, development, and function of various tissue hypersensitivity responses.

Cutaneous lesions elicited in guinea pigs by primary and secondary feeding populations of the argasid tick, Ornithodorus tartakovskyi, were analyzed by light and electron microscopy. Small clusters of basophils appeared at primary bite sites within 24 hr of tick attachment, and by 72 hr constituted approximately 11% of the total leukocytes. Secondary feeding sites exhibited an augmented cellular infiltrate that was dominated by basophils at all times (48-56% of total cells). Eosinophil proliferation was minimal, however, and the remaining cells were of the mononuclear type. Despite mounting a strong cutaneous basophil response of the kind that mediates immune rejection of prolonged-feeding ixodid ticks, the guinea pigs showed no resistance to the fast-feeding Argasidae. It is suggested that argasid ticks probably complete their blood meal prior to basophil arrival at the bite site. Electron microscopy indicated that the number of epidermal Langerhans cells increased with time in both primary and secondary lesions; these cells were more numerous in challenge infections however, and seemed also to occur in the dermis. Basophils at secondary bite sites exhibited three kinds of structural alterations classified as: (1) piecemeal alterations--involving a vesicular degranulation mechanism; (2) an anaphylactic-type of alteration--involving single or compound exocytosis of whole granules; and (3) cytotoxic alterations culminating in complete disintegration. The majority of basophils in 72 hr secondary lesions exhibited cytotoxic alterations. It is suggested that such changes result from contact with tick-derived toxins or enzymes.

Hapten-specific delayed time course skin reactions containing predominant accumulations of basophils and eosinophils were elicited in newborn guinea pigs after i.v. transfer of small amounts of oxazolone immune serum. The immune serum was fractionated by column chromatography procedures, and the fractions were examined for their ability in transferring this form of cutaneous basophil hypersensitivity (CBH). Only the 7S IgG-containing peak from Sephadex G-200 columns, and only the IgG1-containing fractions from DEAE columns, transferred CBH. An affinity column of bound oxazolone removed the activity from immune serum, and it could be recovered from the column by eluting with soluble oxazolone. About 35 microgram of purified IgG1 anti-oxazolone antibody could systemically transfer CBH reactivity. An immunoadsorbant column of anti-IgG1 removed this activity, but a column of anti-IgG2 did not. None of the procedures were able to separate activity in transferring CBH from passive cutaneous anaphylactic (PCA) activity classically associated with guinea pig IgG1 antibody. IgG1 from 8-day immune and 31-day hyperimmune donors were both effective. The average association constant of 8-day antibody was 8 X 10(-4) M-1. Transfer of cutaneous basophil reactions can be mediated by low affinity serum 7S IgG1 antibody.

A previous report showed that infection with the nematode Nippostrongylus brasiliensis stimulates a basophilia as well as an eosinophilia in the blood of August rats. The present study shows that blood levels of basophils and eosinophils were increased in two other rat strains, one inbred and one outbred, after infection with N. brasiliensis, and infection of two inbred rat strains with Trichinella spiralis also stimulated a basophilia as well as an eosinophilia. No increase occurred in basophils or eosinophils in athymic (nude) rats infected with N. brasiliensis, although both these cell types were found in the blood of control, specific pathogen free, nude rats in numbers comparable to those in specific pathogen free, heterozygote controls of the same strain. Rat basophils usually have few granules and in blood smears often appear as if they were partly degranulated. Basophils from uninfected nude rats contained more negative than positive staining granules compared with basophils from parasitized heterozygotes. The possession of small numbers of granules which vary in their reaction to stains of the Romanowski type is a normal feature of rat basophils in blood smears. Consequently rat basophils differ in these respects from those of other species.

Contact hypersensitivity reactions were elicited by intraluminal application of DNCB to the colon of guinea pigs and were found sometimes to contain large infiltrates of basophils. These colonic basophil hypersensitivity reactions were accompanied by systemic sensitization and were not seen in nonspecific colonic inflammatory responses induced by croton oil. The heterogenous immune inflammatory responses induced in the colon by DNCB contact sensitivity may serve as a model for the study of inflammatory bowel disease.

Cellular transfer of cutaneous basophil hypersensitivity (CBH) was studied. Guinea pigs immunized for CBH with incomplete Freund's adjuvant (IFA) provided cells which could transfer delayed and basophil-rich reactions in skin tests of recipients. Guinea pigs immunized with complete classical tuberculin-type delayed hypersensitivity reactions (DH), which are characteristically devoid of basophils. However, recipients of cells from donors with DH, surprisingly, were found to have delayed skin reactions containing large basophil infiltrates which were lacking in the donors. Thus, recipients of classical cell transfers of tuberculin-type DH had delayed reactions which resembled CBH. Control experiments verified that the cell transfer of CBH from donors with DH was due to passive transfer with live cells and not transfer of contaminating humoral factors or active sensitization of recipients. It was concluded that cutaneous basophil responses were suppressed in CFA-immunized donors and expressed in cell transfer recipients. Cells from donors immunized with CFA were enriched for nonadherent and nonimmunoglobulin-bearing lymphocytes by passage through nylon wool columns, and these cells transferred conjugate specific CBH reactions. It was concluded that cells mediating these transfers were probably T cells. The finding of basophils in cell transfers of DH and a variety of other findings suggesting complex regulation of basophil numbers in tissue lead to the conclusion that the term CBH be used to simply describe a basophil-containing skin reaction.

Resistance to infestation by larval Amblyomma americanum or Rhipicephalus sanguineus ticks was transferred to naive guinea pigs with peritoneal exudate cells (PEC) or serum from donors immunized by prior infestation with homologous tick larvae. In the A. americanum system, PEC transfer induced 87% tick rejection, which was similar to the level of resistance in actively sensitized hosts. In the R. sanguineus system, PEC conferred resistance (39% rejection) that was weaker than in actively sensitized hosts (57% rejection). In both systems, immune serum conferred significant but weaker resistance (20 to 29% rejection). In actively sensitized hosts, resistance to each tick species was specific, but there was considerable cross-reactive resistance. Basophils dominated the 24-hr challenge feeding sites of A. americanum ticks in actively sensitized hosts (69% of the infiltrate) and recipients of sensitized PEC (69%). Mononuclear cells were dominant (69% of the infiltrate) in the challenged tissues of immune serum recipients that had a significant but weaker cutaneous basophil response (24%). Mononuclear cells also dominated (58% of the infiltrate) the 24-hr challenge feeding sites of R. sanguineus ticks in actively sensitized hosts, but there were also 24% basophils. These studies demonstrate that immune resistance to tick is dependent on sensitized lymphoid cells or serum components, and that sensitized cells or serum can transfer a cutaneous basophil response that is associated with immune resistance. Rejection of ticks is usually associated with large basophil infiltrates, but sometimes mononuclear cells are dominant. Thus, immune resistance of guinea pigs to ticks is a heterogeneous response in which immune cells and serum probably act to recruit diverse effector leukocytes to mediate rejection that is specific but significantly cross-reactive.

Cutaneous basophil hypersensitivity, an immune inflammatory reaction characterized by infiltrates of basophils and a delayed time-course, was studied in guinea pigs contact sensitized with oxazolone. Routine histological techniques, employing ordinary paraffin sections, were modified to study this reaction. When biopsies of contact lesions were processed by these methods dense infiltrates of basophils could be demonstrated. Animals sensitized with complete Freund's adjuvant emulsified with oxazolone-keyhole limpet hemocyanin conjugates also developed delayed-in-time responses to contact challenge with oxazolone but not to picryl chloride. These hapten-specific delayed-in-time reactions also contained substantial numbers of basophils. Transfer of serum from actively sensitized guinea pigs resulted in specific accumulation of basophils at challenge sites of recipients. Thus, in this experimental system, cutaneous basophil hypersensitivity was found to be a hapten-specific delayed-in-time reaction that could be transferred with immune serum.