The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes.

The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

Inhibition of DNA synthesis by a small-cell lung carcinoma-derived protein.

A tumor-derived factor that inhibits cellular DNA synthesis was identified. The factor was extractable from a small-cell lung carcinoma cell line grown in either chemically defined medium or nu/nu mice and inhibited tritiated thymidine ([3H]dThd) incorporation by tumor cell lines of autologous, allogeneic, and xenogeneic origins. The viability of nonproliferating cells from normal tissue was not affected. Tumor extract inhibitory activity was trypsin labile but was resistant to other proteases, neuraminidase, lipase, DNase, RNase, glucosidase, extremes of pH-temperature, and reducing conditions. Inhibitory activity was reversibly bound to helix pomatia lectin but not to lentil, wheat germ, or concanavalin A lectins. Purification by size-exclusion high-performance liquid chromatography yielded a bioactive unimodal 12-kilodalton (kd) peak. The bioactive 12-kd moiety could be eluted from sodium dodecyl sulfate-polyacrylamide gels. Redosing of populations of the T-lymphoblastoid cell line CEM achieved an early (24 hr) sustained depression of pulse [3H]dThd incorporation and ultimately led to decreased population density of factor-treated populations. DNA histogram analysis demonstrated no change in cell cycle phase distribution after factor treatment. 5-Bromo-2'-deoxyuridine (BrdUrd) vs. propidium iodide with the two-parameter Fluorescence-Activated Cell Sorter analysis showed relative inhibition of non-S-phase BrdUrd uptake at 24 hours. A cell-free DNA polymerase assay demonstrated significant inhibition of non-alpha-polymerase-associated DNA synthesis in factor-treated cells. These studies suggest that this tumor-derived inhibitor of DNA synthesis represents a class of cellular products involved in the autoregulation of growth by regulation of DNA synthetic activity.

In vitro studies of cell-mediated immunity to Moloney murine leukemia virus and Moloney leukemia-associated surface antigens.

Cell-mediated immunity to Moloney murine leukemia virus (M-MuLV) and to tumor-associated surface antigens of leukemia cells induced by the virus was studied with an in vitro migration inhibition factor assay. Spleen cells of C57BL/6N mice at Day 14 following inoculation with Moloney murine sarcoma virus, produced migration inhibition factor in response to M-MuLV. The Moloney murine sarcoma virus-immune spleen cells, however, did not respond to other murine type C viruses, to AKR and Rauscher viruses, or to murine mammary tumor virus. The immune spleen cells also responded specifically to purified glycoprotein with molecular weights of 69,000 and 71,000 and proteins with molecular weights of 30,000 and 12,000, but not to protein with a molecular weight of 10,000, of the homologous M-MuLV. Migration inhibition factor production was also observed in response to soluble 3 M KCl extracts of leukemia cells, MBL-2, induced by M-MuLV. Similarly, the immune spleen cells responded to membrane fractions purified from the MBL-2 cells. Comparable membrane fractions prepared from a Gross virus-induced leukemia, E male G2, and a radiation-induced leukemia, RL male 1, were not active. The tumor-associated surface antigens of MBL-2 membranes could be solubilized by the detergent, Nonident P-40. Thus, C57BL/6N mice inoculated with Moloney murine sarcoma virus developed cell-mediated immunity to envelope and some internal antigens of M-MuLV and also to tumor-associated surface antigens of a tumor induced by this leukemia virus.

Mediation of reduction of spontaneous and experimental pulmonary metastases by ricin A-chain immunotoxin 45-2D9-RTA with potentiation by systemic monensin in mice.

We developed a model to assess the therapeutic effects of the 45-2D9-ricin A-chain immunotoxin (RTA) on pulmonary metastases. The 45-2D9 mouse monoclonal antibody recognizes a Mr 74,000 glycoprotein highly expressed by rat fibroblasts transformed with the Kirsten sarcoma virus (transformed rat fibroblasts). These cells metastasize spontaneously and form lung colonies in nu/nu and irradiated BALB/c mice. Injection i.v. of 45-2D9-RTA specifically reduced formation of spontaneous pulmonary metastases and lung colonies originating from freshly disaggregated tumor cells or cultured cells. Antibody alone or mixed with unconjugated ricin A chain and an immunotoxin that recognizes a melanoma-associated antigen were ineffective. Unconjugated 45-2D9 antibody specifically blocked the 45-2D9-RTA activity in vivo. Administration of the lysosomotrophic agents ammonium chloride and chloroquine in vivo did not potentiate immunotoxin-mediated reduction in lung colonies although they were effective in vitro. Monensin potentiated 45-2D9-RTA activity in vitro and in vivo.

Analysis of expression of cell surface antigen Mr 74,000 phosphoglycoprotein in normal, oncogene-transformed, and neoplastic rat cell lines.

A Mr 74,000 phosphoglycoprotein (gp74) present on the surface of oncogene-transformed murine cells but not untransformed NIH 3T3 cells was previously identified with mouse monoclonal antibody 45-2D9. The original cell population used as the immunogen was found to consist of two cell populations. The purpose of this study was to characterize these cell populations; determine the distribution of gp74 on normal, transformed, and neoplastic cells; and to characterize the gp74 molecule. Southern hybridization studies of cloned cell populations demonstrated that the immunizing cell population consisted of c-Ha-ras-transfected NIH 3T3 cells and Kirsten sarcoma virus-transformed rat cells (TRF cells). TRF cells showed a high level of gp74 expression. We observed that the expression of gp74 was increased on chemically and spontaneously transformed rat cells compared to untransformed rat cells. No binding of monoclonal antibody 45-2D9 was detected to rat adult and fetal tissue. Immunoperoxidase staining, immunofluorescence flow cytometry, and immunoprecipitation analysis of dimethylbenz[a]anthracene-induced metastatic 13762NF rat mammary adenocarcinoma clonal sublines demonstrated an inverse relationship between gp74 expression and metastatic phenotype. gp74 was immunoprecipitated from two low and medium metastatic clonal sublines (MTC and MTF7), but not from highly metastatic clone MTLn3 cells. Biosynthetic labeling and immunoprecipitation studies demonstrated that gp74 was phosphorylated on serine residues and was not secreted from transformed cells. No detectable protein kinase activity in an immune complex assay was associated with this molecule. We conclude that increased gp74 expression by rat cells is associated with transformed and neoplastic cells.

In Xenopus oocytes the human C3a and C5a receptors elicit a promiscuous response to the anaphylatoxins.

The Xenopus laevis oocyte has been widely utilized for cloning and functional expression of G-protein coupled receptors (GPCR). This system was used for the functional expression and characterization of the recently identified human C3a receptor. Complementary RNA from the human C3a receptor was transcribed in vitro and microinjected into Xenopus oocytes for functional characterization. A positive response to a synthetic C3a peptide agonist and to C3a, but not to platelet activating factor or fMetLeuPhe was detected. In addition, a response of approximately one third the amplitude obtained with C3a was obtained with rC5a. Conversely, oocytes co-injected with the C5a receptor and total RNA isolated from U937 cells responded to C5a as well as to C3a and the C3a synthetic peptide. A functional response with the anaphylatoxin C3a receptor in oocytes was dependent on co-injection of a pertussis toxin sensitive complementary human factor which could be supplied by co-injection of total RNA isolated from U937 cells. Oocytes expressing the anaphylatoxin C3a and C5a receptors responded to both agonists, in each case the response to the cognate ligand was substantially more robust than the response elicited by the other anaphylatoxin.

Cloning and characterization of a rabbit ortholog of human Galpha16 and mouse G(alpha)15.

A cDNA was cloned from a rabbit spleen cDNA library which encoded a G-protein alpha subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Galpha16 and mouse G(alpha)15, respectively. The rabbit G(alpha)subunit cDNA was subcloned into a mammalian expression vector and transiently co-transfected into HEK-293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G alpha subunit behaved similarly to G(alpha)15 or G(alpha)16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit G(alpha) subunit appears to be the ortholog of human G(alpha)16 and mouse G(alpha)15.

Conversion of murine Fabs isolated from a combinatorial phage display library to full length immunoglobulins.

The use of combinatorial Ig libraries displayed on the surface of bacteriophage has advantages over traditional hybridoma techniques for the generation of mAbs but in many instances full length Igs may be more desirable than Fab fragments. Two murine Fabs reactive with the human complement component C5a, recovered from a combinatorial library, were converted to full length IgG2a mAbs. The VH and VL domains of these antibodies were removed from the bacterial expression vector used for the combinatorial library construction, and subcloned into individual mammalian expression vectors containing the corresponding Ig heavy and light chain constant regions. The subcloning relied on 5' restriction endonuclease sites encoded by the oligonucleotide primers originally used to amplify the Ig cDNAs and 3' sites conserved in CH1 and C kappa. These vectors were co-transfected into COS cells yielding full length IgG2a versions of the anti-C5a antibodies. The mAbs, purified from the culture supernatant, retained the full activity of the Fabs, binding specifically to and neutralizing human recombinant C5a. Refined versions of the mammalian expression vectors have been constructed for single step conversion of murine recombinant Fabs, recovered from combinatorial libraries, to IgG2a mAbs.

Differential vasoconstrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey.

1. Urotensin-II (U-II) and its G-protein-coupled receptor, GPR14, are expressed within mammalian cardiac and peripheral vascular tissue and, as such, may regulate mammalian cardiovascular function. The present study details the vasoconstrictor profile of this cyclic undecapeptide in different vascular tissues isolated from a diverse range of mammalian species (rats, mice, dogs, pigs, marmosets and cynomolgus monkeys). 2. The vasoconstrictor activity of human U-II was dependent upon the anatomical origin of the vessel studied and the species from which it was isolated. In the rat, constrictor responses were most pronounced in thoracic aortae and carotid arteries: -log[EC(50)]s 9.09+/-0.19 and 8.84+/-0.21, R(max)s 143+/-21 and 67+/-26% 60 mM KCl, respectively (compared, for example, to -log[EC(50)] 7.90+/-0.11 and R(max) 142+/-12% 60 mM KCl for endothelin-1 [ET-1] in thoracic aortae). Responses were, however, absent in mice aortae (-log[EC(50)] <6.50). These findings were further contrasted by the observation that U-II was a 'coronary-selective' spasmogen in the dog (-log[EC(50)] 9.46+/-0.11, R(max) 109+/-23% 60 mM KCl in LCX coronary artery), yet exhibited a broad spectrum of vasoconstrictor activity in arterial tissue from Old World monkeys (-log[EC(50)]s range from 8.96+/-0.15 to 9.92+/-0.13, R(max)s from 43+/-16 to 527+/-135% 60 mM KCl). Interestingly, significant differences in reproducibility and vasoconstrictor efficacy were seen in tissue from pigs and New World primates (vessels which responded to noradrenaline, phenylephrine, KCl or ET-1 consistently). 3. Thus, human U-II is a potent, efficacious vasoconstrictor of a variety of mammalian vascular tissues. Although significant species/anatomical variations exist, the data support the hypothesis that U-II influences the physiological regulation of mammalian cardiovascular function.

Molecular and pharmacological characterization of the murine tachykinin NK(3) receptor.

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.

Immunoregulatory factors derived from human tumors. II. Partial purification and further immunobiochemical characterization of a human sarcoma-derived immunosuppressive factor expressing HLA-DR and immunoglobulin-related determinants.

An immunoregulatory factor (IRF) that suppresses Con A-mediated peripheral blood mononuclear cell (PBM) proliferative responses was partially purified by DEAE anion exchange chromatography and affinity chromatography from a 3 M KCl extract of a human liposarcoma. The factor (m.w. = 70K) co-purified with albumin, monitored by two-dimensional gel electrophoresis, and demonstrated a heterogeneous isoelectric point (pI 7.6-7.8). Xenoantisera produced against the DEAE-purified fraction and coupled to Affigel 10 removed suppressive activity that could subsequently be eluted by glycine-HCl, pH 3.5. An anti-albumin column partially removed activity, but the unbound 70K factor could still be detected in the column effluent. A xenoantiserum to this 70K effluent coupled to acrylamide beads completely removed the immunosuppressive activity in immunodepletion experiments. Further direct binding enzyme-linked immunoassays (ELISA) and solid-phase immunoabsorption experiments with monoclonal antibodies to human anti-HLA-DR framework determinants and a constant region of the IgM mu-chain demonstrated determinants on the 70K factor recognized by these antibodies.

The mouse anaphylatoxin C3a receptor: molecular cloning, genomic organization, and functional expression.

The anaphylatoxin C3a receptor (C3aR) is unique among the family of G protein-coupled receptors in possessing an unusually large predicted second extracellular loop. To isolate the mouse C3aR, a probe derived from this extracellular loop was used to screen a mouse brain cDNA library. A 3.3-kb cDNA encoding an open reading frame of 477 amino acids was identified. The predicted amino acid contained four predicted N-linked glycosylation sites and was 65% identical to the 482 amino acids comprising the coding region of the human C3aR. Northern blot analysis revealed that this gene was expressed in a variety of mouse tissue and was especially abundant in heart and lung tissues. The mouse C3aR cDNA was used as a probe to isolate a mouse C3aR genomic clone. The nucleotide sequence of the mouse C3aR genomic clone was identical to the cDNA throughout the coding region, indicating that the receptor is encoded on a single exon. The C3aR cDNA was subcloned into a mammalian expression vector and transiently expressed in HEK-293 cells. Binding of radiolabeled C3a to the transfected cells was competed in a dose-dependent manner by increasing concentrations of unlabeled C3a, with a 50% inhibiting concentration of 10 nM. Similar to the human C3aR, RBL-2H3 rat basophilic cells stably expressing this receptor responded in a dose-dependent manner to C3a, a synthetic C3a peptide agonist, but not C4a or C5a, with a vigorous calcium mobilization.

Isolation of neutralizing anti-C5a monoclonal antibodies from a filamentous phage monovalent Fab display library.

A panel of mAbs against the activated complement component C5a was obtained from a filamentous phage M13-Fab display library generated from mice immunized with human rC5a. Fabs isolated from the library after iterative selection vs rC5a bound to both rC5a and purified C5. To isolate Fabs specific for neoepitopes expressed on C5a but not on the native complement component C5 the library was rescreened in a competitive manner. The phage Fab library was first incubated with immobilized C5 to deplete C5 reactive Fabs. The C5 nonadherent phage were then incubated with immobilized rC5a in the presence of soluble C5. Bound phage were eluted and subjected to two additional cycles of subtraction with immobilized C5 and selection with immobilized rC5a in the presence of soluble C5. After three cycles of this competitive biopanning four Fabs reactive with rC5a were isolated. Two bound preferentially with and neutralized C5a. Competitive biopanning of phage display libraries may increase the probability of identification of Abs of the desired specificity.

Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins.

Tumor stasis factor (TSF): a possible mechanism for the regulation of tumor cell proliferation.

A new class of factors that regulates tumor cell division in vitro can be isolated from fresh and cultured tumor cells by 3 M KCl extraction. Tumor stasis factors (TSF) inhibiting cultured tumor cell proliferation were extracted from 8 of 11 fresh human tumors and 2 cultured tumor cell lines. TSF inhibited [3H]Tdr incorporation by allogeneic and autologous cultured tumor cells in a dose-dependent manner. Extracts of normal human tissues and benign tumors did not demonstrate inhibition with the exception of liver. The mechanism of inhibition was cytostatic and not cytotoxic as demonstrated with trypan blue exclusion by tumor cells following TSF treatment, maintenance of intact tumor cell monolayers following addition of TSF, and lack of inhibition of Con A-mediated lymphocyte proliferation by TSF. TSF activity could be reversed by washing for up to 48 hr of incubation and was resistant to heat, pH alterations, reducing agents, proteases, and glycosidases. However, the active moiety bound to lentil lectin and could be purified 80-fold by preparative isoelectric focusing. These factors may represent a novel regulatory mechanism for tumor cell proliferation.

Monoclonal antibody 45-2D9 conjugated to the A chain of ricin is specifically toxic to c-Ha-ras-transfected NIH 3T3 cells expressing gp74.

Monoclonal antibody 45-2D9 recognizes a 74K Mr glycoprotein determinant on a c-Ha-ras oncogene-transfected cell line (45-342). An immunotoxin was made by conjugating this antibody to the A chain of ricin toxin (RTA). The immunotoxin could mediate essentially complete inhibition of leucine and thymidine incorporation by 45-342 cells prepared as single cell suspensions from tumors grown in vivo. Addition of ammonium chloride to the culture medium potentiated this inhibition, but the magnitude of this effect was dependent on incubation time and cell concentration. The immunotoxin effects were noted at concentrations 100-fold lower than similar effects caused by unconjugated RTA, and the 45-2D9 antibody had no effect in the assay system. Immunotoxins directed against antigens not expressed by 45-342 were not effective, and the 45-2D9 immunotoxin was not specifically toxic to other transfected cells not expressing the gp74 antigen. After a 72-hr incubation, lysis of 80% of the 45-342 cells was demonstrated by trypan blue exclusion. Complete inhibition of 45-342 colony formation was achieved at 10 days with a 10(-9) M concentration of the specific immunotoxin. These results indicate that an immunotoxin with specific reactivity towards an oncogene-transformed cell can be made, and that such cells derived from fresh tumors are susceptible to immunoconjugate-mediated toxicity.

Monoclonal antibody 45-2D9 recognizes a cell surface glycoprotein on a human c-Ha-ras transformed cell line (45-342) and a shared epitope on human tumors.

A monoclonal antibody (45-2D9) produced after immunization of BALB/c mice with the c-Ha-ras NIH 3T3 tertiary transfectant (45-342) recognized a determinant expressed by the primary, three of three secondary, and one of three tertiary transfectants, but not by NIH 3T3 cells. The determinant was present on the cell surface and was distinct from murine leukemia virus gp70 by absorption studies. Biosynthetic labeling and immunoprecipitation studies with [35S]methionine and [3H]glucosamine demonstrated that 45-2D9 recognizes a 74,000 Mr glycoprotein with minor bands of 90,000 and 180,000 Mr on SDS-PAGE. Pulse chase studies demonstrated a 68,000 Mr precursor molecule that incorporated only [35S]methionine. The distribution of the epitope recognized by 45-2D9 was assessed by immunoperoxidase staining. The antigen was not detected on 10 primary and metastatic murine tumors or 11 transformed murine cell lines. However, a variety of surgically excised human tumors demonstrated intense staining, whereas staining of normal tissues was minimal or not detectable. Thus a human oncogene-transfected cell can express a new cell surface determinant apparently unrelated to the oncogene product, which is also selectively expressed by human tumors.

Receptor for the C3a anaphylatoxin is expressed by neurons and glial cells.

Little is known about the expression of the receptor for complement anaphylatoxin C3a (C3aR) in the central nervous system (CNS). In this study, we provide the first evidence that neurons are the predominant cell type expressing C3aR in the normal CNS. By using in situ hybridization (ISH) and immunohistochemistry, we found that C3aR is constitutively expressed at high levels in cortical and hippocampal neurons as well as in Purkinje cells. Moreover, we showed that primary culture of human astrocytes and microglia express the C3aR mRNA as assessed by RT-PCR. In situ hybridization performed on rat primary astrocytes confirmed the RT-PCR result demonstrating C3aR expression by astrocytes. In experimental allergic encephalitis (EAE), C3aR expression was elevated on microglia, infiltrating monocyte-macrophage cells and a few astrocytes, whereas neuronal expression remained unchanged during the course of the disease. These data demonstrate that the C3aR is expressed primarily by neurons in the normal CNS and that its neuronal expression is not dramatically upregulated under inflammation. This is in contrast to the increased neuronal expression of the C5aR in several inflammatory CNS conditions. The high constitutive expression of the C3aR by neurons suggests this receptor may play an important role in normal physiological conditions in the CNS.

Human T cells express the C5a receptor and are chemoattracted to C5a.

The anaphylatoxin C5a is a potent mediator of inflammation that exerts a broad range of activity on cells of the myeloid lineage. In this study, we present the first evidence that human T cells express the C5a receptor (C5aR) and are chemotactic to C5a. Using FACS analysis, we found that the C5aR was expressed at a low basal level on unstimulated T cells and was strikingly up-regulated upon PHA stimulation in a time- and dose-dependent manner. CD3+ sorted T cells as well as Jurkat T cells were shown to express C5aR mRNA as assessed by RT-PCR. Moreover, semiquantitative RT-PCR analysis demonstrated that C5aR mRNA was down-regulated in purified T cells upon long-term PHA stimulation. To demonstrate that C5a was biologically active on T cells, we investigated the chemotactic activity of C5a and observed that purified CD3+ T cells are chemotactic to C5a at nanomolar concentrations. Finally, using a combination of in situ hybridization and immunohistochemistry, we showed that the T cells infiltrating the central nervous system during experimental allergic encephalomyelitis express the C5aR mRNA. In summary, these results suggest that C5a exerts direct effects on T cells and could be involved in the trafficking of T cells under physiological and pathological conditions, including inflammatory diseases of the central nervous system.

A large scale method of separating multiple lymphokines secreted by the murine EL-4 thymoma.

The in vivo administration of lymphokines is a new approach to immunotherapy with possible applications to the treatment of tumors and infectious processes. These lymphokines are produced in very small quantities and purification of these molecules has proven difficult. A rapid two step method was used to isolate different lymphokines from large quantities of conditioned media produced by the murine EL-4 cell line. Interleukin-2 (IL-2), colony stimulating factor (CSF) and macrophage activating factor (MAF) are lymphokines with distinct biologic activities and are secreted by the EL-4 cell line. Their isolation involved initial batch purification on trimethylsilyl-controlled pore glass beads followed by reversed phase high pressure liquid chromatography. Recovery of IL-2 activity was greater than 100% of initial activity. The specific activity of the purified IL-2 ranged from 2 X 10(6) to 3.6 X 10(7) U/mg protein. Colony stimulating factor (CSF) and macrophage activating factor (MAF) were also separated on the chromatographic columns. This technique is useful for the purification of large quantities of these lymphokines which have potential in vivo applications.