Lymphatic vessel growth, or lymphangiogenesis, is regulated by vascular endothelial growth factor-C (VEGF-C) and -D via VEGF receptor 3 (VEGFR-3). Recent studies suggest that VEGF, which does not bind to VEGFR-3, can also induce lymphangiogenesis through unknown mechanisms. To dissect the receptor pathway that triggers VEGFR-3–independent lymphangiogenesis, we used both transgenic and adenoviral overexpression of placenta growth factor (PlGF) and VEGF-E, which are specific activators of VEGFR-1 and -2, respectively. Unlike PlGF, VEGF-E induced circumferential lymphatic vessel hyperplasia, but essentially no new vessel sprouting, when transduced into mouse skin via adenoviral vectors. This effect was not inhibited by blocking VEGF-C and -D. Postnatal lymphatic hyperplasia, without increased density of lymphatic vessels, was also detected in transgenic mice expressing VEGF-E in the skin, but not in mice expressing PlGF. Surprisingly, VEGF-E induced lymphatic hyperplasia postnatally, and it did not rescue the loss of lymphatic vessels in transgenic embryos where VEGF-C and VEGF-D were blocked. Our data suggests that VEGFR-2 signals promote lymphatic vessel enlargement, but unlike in the blood vessels, are not involved in vessel sprouting to generate new lymphatic vessels in vivo.

Vascular endothelial growth factor (VEGF)-A has essential roles in vasculogenesis and angiogenesis, but the downstream steps and mechanisms by which human VEGF-A acts are incompletely understood. We report here that human VEGF-A exerts much of its angiogenic activity by up-regulating the expression of TR3 (mouse homologue Nur77), an immediate-early response gene and orphan nuclear receptor transcription factor previously implicated in tumor cell, lymphocyte, and neuronal growth and apoptosis. Overexpression of TR3 in human umbilical vein endothelial cells (HUVECs) resulted in VEGF-A–independent proliferation, survival, and induction of several cell cycle genes, whereas expression of antisense TR3 abrogated the response to VEGF-A in these assays and also inhibited tube formation. Nur77 was highly expressed in several types of VEGF-A–dependent pathological angiogenesis in vivo. Also, using a novel endothelial cell-selective retroviral targeting system, overexpression of Nur77 DNA potently induced angiogenesis in the absence of exogenous VEGF-A, whereas Nur77 antisense strongly inhibited VEGF-A–induced angiogenesis. B16F1 melanoma growth and angiogenesis were greatly inhibited in Nur77^−/− mice. Mechanistic studies with TR3/Nur77 mutants revealed that TR3/Nur77 exerted most of its effects on cultured HUVECs and its pro-angiogenic effects in vivo, through its transactivation and DNA binding domains (i.e., through transcriptional activity).

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A¹⁶⁴, we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A–expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A¹⁶⁴ also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new “giant” lymphatics generated by VEGF-A¹⁶⁴ were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.

Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcε receptor I (FcεRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the FcεRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of FcεRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.

Circulating leukocytes are thought to extravasate from venules through open interendothelial junctions. To test this paradigm, we injected N-formyl-methionyl-leucyl-phenylalanine (FMLP) intradermally in guinea pigs, harvesting tissue at 5–60 min. At FMLP-injected sites, venular endothelium developed increased surface wrinkling and variation in thickness. Marginating neutrophils formed contacts with endothelial cells and with other neutrophils, sometimes forming chains of linked leukocytes. Adherent neutrophils projected cytoplasmic processes into the underlying endothelium, especially at points of endothelial thinning. To determine the pathway by which neutrophils transmigrated endothelium, we prepared 27 sets of serial electron microscopic sections. Eleven of these encompassed in their entirety openings through which individual neutrophils traversed venular endothelium; in 10 of the 11 sets, neutrophils followed an entirely transendothelial cell course unrelated to interendothelial junctions, findings that were confirmed by computer-assisted three-dimensional reconstructions. Having crossed endothelium, neutrophils often paused before crossing the basal lamina and underlying pericytes that they also commonly traversed by a transcellular pathway. Thus, in response to FMLP, neutrophils emigrated from cutaneous venules by a transcellular route through both endothelial cells and pericytes. It remains to be determined whether these results can be extended to other inflammatory cells or stimuli or to other vascular beds.

The expression of delayed-type hypersensitivity in animals has been inhibited by a variety of anticoagulants, but direct evidence for activation of clotting in the evolution of these reactions has been lacking. Using the fluorescent antibody technique we here demonstrate that fibrin deposition is a prominent and consistent feature of both allergic contact dermatitis and classic delayed hypersensitivity skin reactions in man. Fib was detected in 55 of 58 delayed reactions studied at the peak of their intensity. The characteristic distribution of Fib—principally in the intervascular portions of the reticular dermis with sparing of vessels and their associated cuffs of mononuclear cells—is unusual and quite different from that described in antibody-mediated lesions in animals or man. Fib was found in vessel walls in only 2 of 94 biopsies studied. With a single exception, deposition of immunoglobulins and complement was not observed. The pathogensis and significance of Fib deposition in these reactions are not yet clear. Fib is ultimately derived from circulating fibrinogen, and its accumulation provides additional evidence for locally increased vascular permeability in delayed hypersensitivity. Polymerization of extravascular fibrinogen could be triggered nonspecifically by dermal elements (e.g., collagen) or by a product of sensitized lymphocytes. The appearance of Fib early in the development of these reactions (4–8 h after epicutaneous test with DNCB) and inhibition studies with anticoagulants together suggest that clotting may have a role in their pathogenesis, possibly by the release of bioactive peptides from fibrinogen/fibrin or by contributing to the induration characteristic of delayed hypersensitivity.

The morphologic events associated with the immunologic rejection by strain 2 guinea pigs of ascites variants of two lines of diethylnitrosamine-induced tumors have been studied by light and electron microscopy. Tumor injection sites in the skin of control animals exhibited clusters of viable, actively mitotic tumor cells along with a modest inflammatory infiltrate composed of lymphocytes, macrophages, neutrophils, and rare basophils. In contrast, similar injections of either tumor line in specifically sensitized guinea pigs elicited typical delayed-type skin reactions associated with tumor cell necrosis and a more extensive inflammatory infiltrate including a selective increase in the number of basophilic leukocytes (12%, line 1, or 23%, line 10, of total inflammatory cells). That basophils may have a role in tumor resistance in vivo is suggested by the close anatomic associations observed between basophils and tumor cells, and by the fact that basophils were the only inflammatory cell to demonstrate a relative increase in frequency in the lesions of sensitized as compared with control animals. Moreover, intraperitoneal injection of line 1 tumor in specifically sensitized animals elicited a striking basophilia within 24 h. Unlike macrophages, basophils did not phagocytose tumor cells but did evidence occasional extrusion of granules and frequently exhibited loss of granule staining density, a change that may be related to release of mediator substances. Electron microscope studies of line 1 tumor rejection in the peritoneal cavities of specifically sensitized guinea pigs demonstrated aggregations of "activated" macrophages, lymphocytes, basophils, and damaged or dead tumor cells. These aggregates, held together by complex interdigitations of macrophage villi, closely resembled those occurring in vitro among peritoneal exudate cells whose migration from capillary tubes was inhibited by migration inhibition factor (MIF). Moreover, cells in these aggregates, as well as macrophages inhibited by MIF in vitro, lacked a normal coating of cell surface material.

The interaction between sensitized lymphocytes and specific antigen occurring in delayed hypersensitivity causes bystander macrophages to undergo a variety of light-microscopic, ultrastructural, and biochemical changes, which are reflected in alterations in cell movement and intercellular contacts. Since such alterations involve functions of the cell periphery, we postulated that metabolic changes in this polysaccharide-rich zone would accompany the expression of delayed hypersensitivity. We here demonstrate that the incorporation of radioactive glucosamine by peritoneal macrophages into TCA-precipitable, membrane-associated material is regularly enhanced when these are cultured in the presence of specific antigen and nonadherent cells (lymphocytes) primed for delayed hypersensitivity. Lymphocytes from unsensitized animals, or from animals immunized so as to form antibody but not delayed hypersensitivity, do not stimulate such incorporation. Antigen-induced glucosamine incorporation is maximal at 2 or 3 days of culture and is not observed earlier; it may be elicited with as little as 0.1 µg/ml PPD, and affords an exceedingly reproducible and sensitive index of delayed hypersensitivity. Radioautographic studies indicate that nearly all plastic adherent cells (90% macrophages) incorporate glucosamine and that grains are concentrated in the regions of the perinuclear zone and cell membrane. Subcellular fractionation indicates that nearly 30% of counts and the highest specific activity are associated with the membrane-rich microsomal fraction; the microsomal distribution of counts increases in both absolute and relative terms when macrophages are cultured in the presence of specific antigen and sensitized lymphocytes. Taken together, these data indicate that a sizable fraction of incorporated glucosamine is localized to the vicinity of the cell periphery but lack sufficient resolution to determine whether this material is associated with the cell membrane itself or with the extramembranous cell coat. This last possibility is of particular interest since we have previously shown that macrophage cell coat material is lost or altered as a consequence of an interaction between sensitized lymphocytes and specific antigen.

A variety of cell-mediated hypersensitivity reactions in experimental animals include a prominent infiltrate of basophilic leukocytes. This form of reactivity has been designated cutaneous basophil hypersensitivity and is favored when sensitization to several types of antigen is accomplished without the use of complete Freund's adjuvant. A similar type of hypersensitivity response was sought in man using morphologic techniques which permit identification of basophilic leukocytes. Eight individuals with allergic contact dermatitis to a variety of allergens were studied and six of these developed typical contact reactions with erythema, edema, and epidermal vesicles. The microscopic findings in 3-day biopsies from these individuals differed significantly from classic descriptions of tuberculin hypersensitivity and showed, in addition to mononuclear cells and the characteristic epidermal changes, a substantial infiltrate of basophilic leukocytes and evidence of altered vascular permeability with vascular compaction, dermal edema, and fibrin deposition. Serial biopsies from one individual permitted analysis of the microscopic pathology as it unfolded at successive intervals after patch test. The initial lesion consisted of perivascular accumulations of lymphocytes; this was followed by an influx of basophils and, subsequently, of eosinophils. These findings associate contact allergy in man with the parallel reactions of cutaneous basophil hypersensitivity in animals and provide further evidence for the heterogeneity of the cellular immune response. The data are consistent with the hypothesis that interaction between sensitized lymphocytes and antigen, at a local test site, is responsible for the attraction of basophils. They also directly implicate the clotting system in delayed-type reactions and suggest the possibility of a synergistic relationship between cellular immunity and reactions mediated by basophil-bound, homocytotrophic antibody.

Lymph node cells from guinea pigs immunized with HSA in complete Freund's adjuvant were grown in cultures containing different concentrations of specific antigen. Stimulation of thymidine incorporation was induced with progressively lower concentrations of HSA at successive intervals after sensitization. Moreover, the intensity of delayed skin reactions and the magnitude of stimulation in vitro increased over the same interval. These events are considered compatible with an evolution of the cellular immune response resulting from the selection of lymphoid cells by decreasing concentrations of antigen in vivo. Cells from animals rendered tolerant to HSA failed to respond to specific antigen in culture. As tolerance waned, stimulation was achieved at high but not low antigen concentrations. Tolerance, measured by cutaneous reactivity or by lymphocyte stimulation, was less readily induced in animals sensitized with adjuvant containing a reduced concentration of mycobacteria. Lymph nodes from these animals contained a large population of cells reactive at high antigen concentration, presumably less susceptible to the toleragenic effect of intravenous antigen. The dissociation of delayed hypersensitivity and antibody formation observed early in the immune response and upon recovery from tolerance has permitted correlation of lymphocyte stimulation with delayed hypersensitivity and cutaneous basophil hypersensitivity respectively.

Antigen-mediated stimulation of thymidine incorporation was demonstrated in lymph node cells from guinea pigs immunized with 100 µg human serum albumin in either Freund's incomplete or Freund's complete adjuvant. Animals receiving HSA in IFA exhibited both cutaneous basophil (Jones-Mote) hypersensitivity and lymphocyte stimulation at 1, but not at 6 wk after immunization. Significant stimulation required ≥ 10 µg HSA/ml of culture. Sensitization with HSA in CFA produced delayed hypersensitivity and permitted lymphocyte stimulation at both 1 and 6 wk. Stimulation was observed with as little as 0.1 µg HSA/ml at the later interval. Administration of 5 mg HSA intravenously at the time of sensitization with 100 µg HSA in IFA reduced but did not eliminate both CBH and lymphocyte stimulation at 1 wk. Antigen-specific inhibition of macrophage migration could be demonstrated with exudates from animals immunized with HSA in CFA, but not with HSA in IFA at 3 wk after sensitization. HSA was cleared from depots of CFA and IFA at similar rates, but significantly more antigen appeared in the plasma and subsequently in the draining lymph nodes following administration in IFA. Conversely, accumulated antigen disappeared more rapidly following CFA immunization.

Delayed onset erythematous skin reactions elicited in guinea pigs early in the course of sensitization with azobenzenearsonate-protein conjugates or with protein antigens in incomplete Freund's adjuvant or in saline were found to have a characteristic morphology which sets them apart from delayed hypersensitivity and the classic antibody mediated reactions. The principle feature was massive dermal infiltration with basophilic leukocytes. Mononuclear cells of several types including activated and small lymphocytes, monocytes, macrophages, and blast cells were also present. Such reactions have in the past been designated Jones-Mote hypersensitivity, but we prefer the descriptive term cutaneous basophil hypersensitivity (CBH) for the reasons given. Occasional basophils extruded their granules, and individual granules, retaining their characteristic ultrastructure, were commonly seen in the interstitium. However, intercellular junctions between endothelial cells were closed except during cell emigration and there was no morphologic evidence of an histamine-like effect. The majority of basophils, moreover, did not degranulate but underwent nuclear pyknosis and cytoplasmic degeneration and were phagocytosed by macrophages. Phagocytosed basophil granules retained their ultrastructure. Skin tests performed at late intervals after sensitization had a different time course and morphology. Animals sensitized with protein antigens in complete Freund's adjuvant developed delayed hypersensitivity; however, reactions elicited in such animals at early (but not late) intervals after sensitization contained a prominent basophil component. We interpret such reactions to be a mixture of delayed hypersensitivity and cutaneous basophil hypersensitivity. The function of the basophil in CBH and its relation to the mononuclear cells which accompany it are unknown, and various possibilities are discussed. We conclude that cutaneous basophil hypersensitivity is a distinct immunologic and morphologic entity, occurring early in the course of sensitization with protein antigens incorporated in any of several vehicles. The mechanism of the reaction is presently unknown, and a general hypothesis to explain its pathogenesis has been proposed.

Jones-Mote reactivity, defined as a delayed-type skin reaction, occurs transiently early in the course of immunization with protein antigens or hapten conjugates with or without the adjuvant effect of tubercle bacilli. The skin reaction is typically a flat, well-circumscribed erythema with little induration beginning at about 6 hr, reaching a peak at 18–24 hr, and fading or gone at 48 hr. Immunogenic carrier requirements for hapten-specific Jones-Mote hypersitivity resemble those of antibody production rather than of classic delayed hypersensitivity. Skin test antigen requirements indicate that the Jones-Mote reaction involves an active stimulatory response rather than combination with preformed antibody, since ABA conjugates of nonimmunogenic D-polymers do not work. Studies with ALS and carrageenan suggest that the lymphocyte is an important contributor to the reaction, but the macrophage is not. Because the reactions studied here are operationally different from those described by Jones and Mote and because they have a characteristic histology, the term "cutaneous basophil hypersensitivity" is proposed.

Normal Dutch rabbit lymph node and spleen minces, lymph node cell suspensions, and residues from lymph node cell suspensions were cultured in Millipore chambers with slices of autologous or homologous (New Zealand) ear skin. for varying time intervals. Lymphoid cells exposed to New Zealand ear skin for more than 4 days were found capable of producing typical "transfer reactions" in the specific New Zealand ear skin donor, similar in every way to reactions produced by cells from lymph nodes sensitized in the intact Dutch animal. Heat-killed cells and cells exposed to New Zealand ear skin for less than 4 days (in chambers or in the intact animal) or to Dutch ear skin for any period of time were incapable of eliciting such reactions. It is concluded that normal lymphoid tissues undergo primary sensitization when exposed to homografts in Millipore chambers for suitable periods of time.