CD40 Ligand (CD154) Stimulation of Macrophages to Produce HIV-1-Suppressive ␤-Chemokines

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 5205–5210, April 1998 Immunology

CD40 ligand (CD154) stimulation of macrophages to produce HIV-1-suppressive ␤-chemokines

RICHARD S. KORNBLUTH*†,KRISTIN KEE*, AND DOUGLAS D. RICHMAN‡

*Departments of Medicine, and of ‡Pathology and Medicine, University of California, San Diego, and Department of Veterans Affairs Medical Center, La Jolla, CA 92093

Edited by Anthony S. Fauci, National Institute of Allergy and Infectious Diseases, and approved February 27, 1998 (received for review February 3, 1998)

ABSTRACT ␤-chemokines play an important role in the colony-stimulating factor (GM-CSF) were unable to induce development of immunologic reactions. Macrophages are ma- macrophages to produce ␤-chemokines. Instead, direct cell– jor ␤-chemokine-producing cells during T-cell directed, de- cell contact with cells expressing CD40L was found to induce layed-type hypersensitivity reactions in tissues, and have been macrophages to produce large amounts of these four ␤-che- reported to be important producers of ␤-chemokines in the mokines. lymph nodes of HIV-1-infected individuals. However, the physiological signals responsible for inducing macrophages to ␤ produce -chemokines have not been established. Two soluble MATERIALS AND METHODS T cell products, interferon-␥ and granulocyte-macrophage colony stimulating factor, were added to cultured macro- Reagents. Phenol-extracted LPS from Escherichia coli phages, but failed to stimulate the production of macrophage ␣ ␤ 0111:B4 (Sigma) was dissolved in ethanol and water (1:1, inflammatory protein-1 and -1 ; regulated upon activation, vol͞vol) at 10 mg͞ml and stored at Ϫ20°C. The following were normal T cell expressed and secreted (RANTES); or monocyte purchased from Genzyme: IFN-␥ (1 ϫ 107͞mg), GM-CSF chemoattractant protein-1. Instead, direct cell–cell contact (1.25 ϫ 107 units͞mg), and murine mAbs against CD40L between macrophages and cells engineered to express CD40L (clone M90) and CD40 (clone M3). Recombinant interleukin (also known as CD154) resulted in the production of large amounts of macrophage inflammatory protein-1␣ and -1␤, (IL)-2 and G418 were from Life Technologies (Gaithersburg, and RANTES (all ligands for CCR5), and monocyte chemoat- MD). OKT3 anti-CD3 mAb was from Ortho Diagnostics. tractant protein-1 (a ligand for CCR2). Supernatants from Unlabeled and phycoerythrin (PE)-labeled mAb against hu- .(CD40L-stimulated macrophages protected CD4؉ T cells from man CD40L (clone 24–31) was from Ancell (Bayport, MN infection by a nonsyncytium-inducing strain of HIV-1 (which PE-labeled mAb against murine CD40L (clone MR1) was uses CCR5 as a coreceptor). These results have implications from PharMingen. The medium for 293 cells consisted of for granulomatous diseases, and conditions such as athero- enriched MEM with Earle’s salts (no. 112–039-101, Quality sclerosis and multiple sclerosis, where CD40L-bearing cells Biologicals, Gaithersburg, MD) containing 2 mM L-glutamine have been found in the macrophage-rich lesions where ␤-che- and 10% heat-inactivated fetal bovine serum (FBS; Summit mokines are being produced. Overall, these findings define a Biotechnology, Ft. Collins, CO) (E10). The medium for pe- pathway linking the specific recognition of antigen by T cells ripheral blood mononuclear cells (PBMC) and monocyte- to the production of ␤-chemokines by macrophages. This derived macrophages (MDM) was RPMI 1640 medium con- pathway may play a role in anti-HIV-1 immunity and the taining 2 mM L-glutamine (BioWhittaker) and 10% FBS development of immunologic reactions or lesions. (R10). MDM Cultures. Venous blood was collected with informed ␤-chemokines are required to induce immunologically impor- consent from healthy donors, and PBMC were prepared by tant cells to migrate from blood into tissue (1–3). Included in centrifugation over Ficoll͞Hypaque. Monocytes were then this family of proteins are the macrophage inflammatory isolated by the fibronectin adherence method and plated in proteins (MIP), 1␣ (MIP-1␣) and 1␤ (MIP-1␤), which were 48-well culture plates at 4 ϫ 105 cells per well in 1.0 ml of RPMI first identified as the products of stimulated macrophages (4), 1640 medium containing 10% fresh, unheated, autologous and RANTES (regulated upon activation, normal T cell serum. After 5–7 days, the medium and any residual nonad- expressed and secreted) and monocyte chemoattractant pro- herent cells were removed, and the MDM monolayers (2 ϫ 105 tein-1 (MCP-1), which were first described as the products of cells per well) were refed with R10 and used 3–7 days later activated T cells (5, 6). A number of microbial agents have when they were Ͼ99% positive for nonspecific esterase (15). ␤ been shown to induce the production of these -chemokines by All culture components were prescreened to select nonacti- macrophages, including bacterial lipopolysaccharide (LPS) (4, vating, low endotoxin lots of media and serum as described 7) and viruses such as HIV-1 (8–10). In addition, macrophages (15). in delayed-typed hypersensitivity reactions produce ␤-chemokines (11–14), but it has not been previously determined how the specific recognition of antigen by T cells could be linked This paper was submitted directly (Track II) to the Proceedings office. to the production of ␤-chemokines by macrophages. In this Abbreviations: CCR, CC chemokine receptor; MIP-1, macrophage study, we found that two well-known soluble macrophage inflammatory protein-1; MCP-1, monocyte chemoattractant pro- ␥ ␥ tein-1; MDM, monocyte-derived macrophages; NSI, nonsyncytium- stimulants, interferon- (IFN- ) and granulocyte-macrophage inducing; PBMC, peripheral blood mononuclear cells; RANTES, regulated upon activation, normal T cell expressed and secreted; PE, ␥ The publication costs of this article were defrayed in part by page charge phycoerythrin; IL, interleukin; LPS, lipopolysaccharide; IFN- ; interferon ␥; GM-CSF, granulocyte-macrophage colony-stimulating factor; payment. This article must therefore be hereby marked ‘‘advertisement’’ in PHA, phytohemagglutinin; SI, syncytium inducing. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed at: Department of © 1998 by The National Academy of Sciences 0027-8424͞98͞955205-6$2.00͞0 Medicine-0679, University of California, San Diego, 9500 Gilman PNAS is available online at http:͞͞www.pnas.org. Drive, La Jolla, CA 92093-0679. e-mail: [email protected].

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Generation of Normal and Mutant CD40L-Expressing 293 Preparation of Plasma Membranes from CD40L-Express- Cells. To clone the cDNA for human CD40L, PBMC were ing 293 Cells. Membranes were prepared from roller bottle stimulated with plate-immobilized anti-CD3 (OKT3) over- cultures of stably transfected 293 cells by ultracentrifugation of night, fed with 10 units͞ml IL-2, and selected for surface a crude membrane preparation over a 35–73% sucrose step CD40L expression by using an anti-CD40L mAb (clone 24–31) gradient as described (18, 19). The resulting membranes were and magnetic beads coated with anti-mouse IgG antibodies washed in PBS by repeated centrifugation at 90,000 ϫ g and (Dynal, Oslo). Two additional weekly cycles of restimulation stored at Ϫ80°C until used. To quantify the membranes, they with anti-CD3 and IL-2 and magnetic bead selection were were first solubilized in 0.1% 3-[(3-cholamidopropyl)dimeth- performed. mRNA was then prepared, reverse-transcribed ylammonio]-1-propanesulfonate (CHAPS) and then assayed into cDNA by using random hexamer primers, and amplified for protein content using the bicinchoninic acid method. by PCR using Pfu polymerase (Stratagene). The PCR primers Stimulation of MDM. To study the influence of soluble for human CD40L (originally designed for a previous gener- factors, cytokines were added to MDM and the supernatant ation of expression vector) were sense, 5Ј-GGGGACTAGTA- media were collected 24 hr later. To study the cell contact GATACCATTTCAACTTTAACACAG-3Ј (containing an pathway of macrophage activation, control 293 cells or CD40L- underlined SpeI site) and antisense, 5Ј-GGGCTCGAGCG- 293 cells were added directly to MDM cultures at a 1:1 GCCGCAGTTCTACATGCCTTGGAGTGTATAAT-3Ј cell-to-cell ratio, and the media were collected 24 hr later. In (containing underlined XhoI and NotI sites). Hot-start PCR some experiments, membranes from the 293 cells were added was performed beginning with 94°C for 2.5 min followed by 45 instead of intact cells. In all cases, the MDM supernatants were Ϫ ␤ cycles of the following program: 94°C for 10 sec, 43°C for 30 stored frozen at 80°C until assayed for -chemokines by ELISA (R & D Systems). All cultures were set up in quadru- sec, and 76°C for 5 min. The product was then digested with Ϯ SpeI and NotI and cloned into the NheI and NotI sites of the plicate and the results are expressed as the mean ( SD). expression vector, pcDNA3.1(ϩ) (Invitrogen) to create the HIV-1 Isolates. HIV-1SF-162 (20) was propagated in PBMC cultures that had been stimulated with 3 ␮g͞ml phytohemag- plasmid, pcDNA3.1-CD40L. The final construct was verified ␮ ␤ by sequencing both DNA strands. Following transfection of glutinin (PHA) and fed with R10 containing 50 M -mer- pcDNA3.1-CD40L into 293 cells and selection in E-10 con- captoethanol and 10 units per ml IL-2. The LAV-1 strain of taining G418 (500 ␮g͞ml) cells were further selected by HIV-1LAI was propagated in CEM cells and titered using MT-2 repeated flow sorting using PE-conjugated mAb 24–31 and cell cultures. The macrophage–tropic strain, HIV-1BaL, was growth for 1 week, for a total of three cycles. The ‘‘control 293 propagated and titered in MDM as described (15). p24 Gag cells’’ used in this study were prepared by transfection with the protein was measured by ELISA (Coulter). Anti-HIV-1 Effects of MDM Supernatants. empty expression vector, pcDNA3.1(ϩ), and selection in The HIV-1 G418. suppressive effects of MDM supernatants were detected es- sentially as described by Paxton et al. (21). Activated CD4- To clone the cDNA for murine CD40L, mRNA from a enriched T cells were prepared by stimulating PBMC with BALB͞c mouse spleen was reverse-transcribed into cDNA by PHA (3 ␮g͞ml) and IL-2 (10 units per ml) in RPMI 1640 using random hexamer primers, and amplified by hemi-nested medium containing 10% unheated autologous serum and 50 PCR using Pfu polymerase. The PCR primers for the first 30 ϩ ␮M 2-mercaptoethanol. Contaminating CD8 T cells were cycles were sense, 5Ј-GGGGAAGCTTGCCTCTGTCCCAT- removed by using anti-CD8-coated magnetic beads (Dynal), TCGTTGGTCAG-3Ј (containing an underlined HindIII site) ϩ and the remaining T cells were typically about 90% CD4 by and antisense, 5Ј-CAGGGCAGGTCCTAACTAACTGACT- ϩ flow cytometry. The CD4 T cells were infected with the TGC-3Ј. A 1:1,000 dilution was made for the second 30 cycle nonsyncytium-inducing (NSI) isolate, HIV-1SF162, at an mul- PCR, which used the same sense primer and an internally tiplicity of infection of 0.01 for 1 hr at room temperature, placed antisense primer, 5Ј-GGGGTCTAGACTGCTG- Ј extensively washed, and distributed into the wells of a 48-well CAGCCTAGGACAGCGCAC-3 (containing an underlined plate at 104͞ml. Supernatants from macrophages cultured with XbaI site). Both PCR reactions were performed by hot-start either control 293 cells, CD40L 293 cells, or LPS (100 ng͞ml) beginning with 94°C for 2.5 min followed by the following were added at a final dilution of 1:20. In some experiments, cycling program: 94°C for 10 sec, 53°C for 30 sec, and 76°C for ␤-chemokines in the supernatants were neutralized by prein- 5 min. The product was then digested with HindIII and XbaI ϩ cubating them for 30 min at room temperature with a mixture and cloned into pcDNA3.1( ) to create the plasmid, of 20 ␮g͞ml each of goat polyclonal anti-MIP-1␣ IgG, anti- pcDNA3.1-mCD40L. The final construct was verified by se- MIP-1␤ mAb, and anti-RANTES mAb (R & D Systems). The quencing both DNA strands. Following transfection of cultures were fed every 3–4 days with medium containing IL-2 pcDNA3.1-mCD40L into 293 cells and selection in E-10 and the respective MDM supernatants and antibodies. On day ␮ ͞ ϩ containing G418 (500 g ml), cells were further selected by 14, the CD4 T cell supernatants were collected and tested for repeated flow sorting using PE-conjugated MR1 and growth p24 antigen. for 1 week, for a total of three cycles. As an additional control for human CD40L-293 cells, cells expressing an inactive mutant of CD40L were prepared. This RESULTS mutant encodes the abnormal T147N protein, which is found T Cell Soluble Factors, IFN-␥ and GM-CSF, Fail to Induce in a subset of patients with X-linked hyper-IgM syndrome ␤-Chemokine Production by Macrophages. To study the reg- (X-HIM) (16). The codon for amino acid 147 in CD40L was ulation of ␤-chemokine production in macrophages, condi- changed from Thr (ACC) to Asn (AAC) by overlapping PCR tions were established in which unstimulated MDM cultured mutagenesis (17) using 5Ј-GCTGAAAAAGGATACTACAA- only in R10 medium failed to produce significant levels of CATGAGCAACAACTTGG-3Ј (mutated codon shown in MIP-1␣, MIP-1␤, or RANTES (Fig. 1B). As expected (4, 7), boldface) and its complementary inverse sequence. The final LPS (100 ng͞ml) strongly induced chemokine production. plasmid, pcDNA3.1-T147N-CD40L, was verified by sequenc- However, IFN-␥ (300 ng͞ml) and GM-CSF (100 ng͞ml), two ing both DNA strands and expressed in 293 cells using G418 classic macrophage-stimulating lymphokines (22), did not in- selection and flow sorting as described to create T147N- duce significant amounts of these ␤-chemokines (Ͻ0.25 ng͞ CD40L-293 cells. When evaluated by flow cytometry using ml). PE-conjugated mAb 24–31, the resulting T147N-CD40L-293 Direct Cell–Cell Contact with CD40L-Expressing Cells cells expressed CD40L protein at a level comparable to the Induces Macrophages to Produce Large Amounts of ␤-Che- CD40L-293 cells shown in Fig. 1A. mokines. We next examined the effects of CD40L, a mem- Downloaded by guest on October 1, 2021 Immunology: Kornbluth et al. Proc. Natl. Acad. Sci. USA 95 (1998) 5207

FIG. 1. CD40L induction of ␤-chemokine production by macrophages. (A) Flow cytometry measurement of CD40L surface expression. Isotype control staining of CD40L-293 cells with PE-conjugated anti-CD8 control mAb (thin solid line). Expression of human CD40L on control 293 cells (thin broken line) and CD40L-293 cells (thick solid line) assessed by using PE-conjugated anti-CD40L mAb 24–31. (B) ␤-chemokine production by macrophages. MDM were cultured in medium alone, with added IFN-␥ or GM-CSF, or with added control 293 cells or CD40L-293 cells. LPS was used as a positive control. The mean chemokine concentrations (picogram per milliliter) of the supernatants from quadruplicate wells 24 hr later are shown (ϮSD). ᮀ, MIP-1␣; o, MIP-1␤; Ⅵ, RANTES. (C) Abrogation of CD40L stimulation by anti-CD40 or anti-CD40L mAbs. In an experiment similar to B, neutralizing mAbs were added at the initiation of the CD40L-293 cell-MDM cocultures. Anti-CD40 mAb blocked Ͼ99% and anti-CD40L mAb blocked 94% of the ␤-chemokine release. (D) Stimulation of macrophages by CD40L-bearing plasma membranes. Acellular preparations of membranes from control 293 cells, 293 cells expressing a nonfunctional mutant of human CD40L (T147N), human CD40L-293 cells (CD40L), and murine CD40L-293 cells (mCD40L) were added to MDM in an experiment similar to B.

brane molecule expressed on the surface of certain activated was necessary because CD40L-293 cell supernatant or CD40L- T cells, which acts as a major regulatory signal in humoral and 293 cells separated from MDM by a porous membrane (Trans- cellular immunity (23–27). Recent reports have indicated that well, Costar) were unable to induce ␤-chemokine production CD40L is also the primary molecule responsible for the (data not shown). cell–cell contact pathway of macrophage activation by T cells Isolated CD40L-Bearing Plasma Membranes Induce Mac- (28–31). By exposing MDM to 293 cells engineered to express rophages to Produce ␤-Chemokines. Because the above ex- CD40L (CD40L-293 cells, Fig. 1A), large amounts of MIP-1␣ periments all used live 293 cells in addition to MDM, it was (15–25 ng͞ml), MIP-1␤ (15–25 ng͞ml), and RANTES (1.5–5 important to consider the possibility that the ␤-chemokines ng͞ml) were generated (Fig. 1B) (ranges given are from 14 might be produced by the 293 cells rather than the MDM. independent experiments by using MDM from 6 different Consequently, acellular plasma membranes were prepared donors). ␤-chemokine production was detectable within 3 hr of from the 293 cells and used to stimulate MDM (250 ␮g͞ml). CD40L stimulation (data not shown). In contrast, control 293 Only membranes prepared from 293 cells expressing human or cells were inactive. As an additional control, 293 cells express- murine CD40L were capable of stimulating MDM to produce ing the T147N mutant form of human CD40L were prepared. MIP-1␣, MIP-1␤, and RANTES. Membranes prepared from This mutation results in a protein that is recognized by several control 293 cells or from T147N-CD40L-293 cells were unable anti-CD40L mAbs yet is nonfunctional because it is found in to stimulate MDM (Fig. 1D). a subset of humans with CD40L deficiency disease, the X- Suppression of NSI HIV-1 Replication in CD4؉ T Cells by linked hyper-IgM (X-HIM) syndrome (16). These T147N- Supernatants from CD40L-Stimulated Macrophages. The CD40L-293 cells were completely inactive in inducing MDM three ␤-chemokines initially selected for study were chosen to produce the three ␤-chemokines (data not shown). because they suppress the replication of NSI strains of HIV-1 To prove that a CD40L͞CD40 interaction was necessary for (33) by binding to CC chemokine receptor 5 (CCR5), the chemokine production, neutralizing mAbs directed against ␤-chemokine receptor that has been shown to be critical for the either CD40L or CD40 were added to the cocultures of acquisition of HIV-1 infection (34–36). To determine whether CD40L-293 cells and MDM (32). Antibody against CD40 (1 the numbers of ␤-chemokines produced by CD40L-stimulated ␮g͞ml) prevented Ͼ99% and antibody against CD40L (10 macrophages were sufficient to inhibit the replication of a virus ␮g͞ml) prevented 94% of the induced production of the that uses CCR5 as a coreceptor, purified CD4ϩ T cells were ␤-chemokines (Fig. 1C). Furthermore, direct cell–cell contact stimulated with PHA and IL-2 and infected with the NSI Downloaded by guest on October 1, 2021 5208 Immunology: Kornbluth et al. Proc. Natl. Acad. Sci. USA 95 (1998)

ϩ isolate, HIV-1SF162. The CD4 T cells were fed twice a week other chemokines of immunologic significance, MCP-1 was for 2 weeks and their supernatants were evaluated for HIV-1 chosen as an additional ␤-chemokine for study. MCP-1 is p24 antigen as a measure of virus replication. As expected, particularly chemotactic for monocytes and has been found in supernatants from MDM cultured only in medium or MDM atherosclerotic plaques (39) and granulomatous lesions (40). cocultured with control 293 cells (both of which contained As with the other three ␤-chemokines examined, CD40L-293 almost none of the three ␤-chemokines (Fig. 1B)) did not cells, but not control cells, stimulated MDM to produce large ϩ affect HIV-1 replication in the CD4 T cells (Fig. 2). However, amounts of MCP-1 (Fig. 3). supernatants from CD40L-stimulated MDM (which contained ␤ high levels of the -chemokines) were highly effective at DISCUSSION preventing the replication of this NSI HIV-1 strain. To determine whether the three ␤-chemokines were responsible for this These results have particular relevance to HIV-1 infection and protection, supernatants from CD40L-stimulated MDM were imply that T cells that express CD40L upon activation could preincubated with neutralizing antibodies against MIP-1␣, play a special role in controlling HIV-1 replication (Fig. 4). MIP-1␤, and RANTES, which eliminated their anti-HIV-1 Immune responses that activate CD4ϩ T cells are known to activity (Fig. 2). make them more vulnerable to HIV-1 infection (41). However, Unlike NSI HIV-1 strains, syncytium-inducing (SI) strains a subset of CD4ϩ T cells can express CD40L on their cell use CXCR4 as a coreceptor rather than CCR5, and their surfaces within a few hours following T cell activation (42). infectivity is not affected by MIP-1␣, MIP-1␤, or RANTES Because the production of ␤-chemokines by MDM is also rapid (33). Instead, SDF-1, an ␣-chemokine that is the ligand for and begins within 3 hr after CD40L stimulation, it is possible ϩ CXCR4, is capable of blocking the infection of CD4 T cells that such CD40LϩCD4ϩ T cells could induce enough chemo- by many SI strains (34–36). To evaluate the effects of CD40L- kine production by macrophages to protect both themselves stimulated MDM supernatants against an SI strain of HIV-1, and any adjacent CD4ϩ T cells from infection by NSI strains ϩ these supernatants were added to CD4 T cells exposed to of HIV-1. [In this context, it should be noted that dendritic HIV-1LAI. Unlike the results with the NSI strain, the CD40L- cells, another but rarer type of antigen-presenting cell, have ϩ stimulated MDM supernatants failed to protect CD4 T cells also been reported to produce large amounts of MIP-1␣ after from infection by this SI virus. Consistent with these results, a CD40L stimulation (43).] Overall, our data indicate that T cells reverse transcriptase-PCR analysis showed only trace expres- (both CD4ϩ and CD8ϩ), which recognize HIV-1 antigens and sion of SDF-1 mRNA in MDM, and this was not upregulated express CD40L, could form an additional ␤-chemokine- by CD40L stimulation (data not shown). Also, consistent with mediated pathway of acquired immunologic resistance to other reports that ␤-chemokines do not protect macrophages HIV-1 infection, along with anti-HIV-1 antibodies, cytotoxic from HIV-1 infection (37, 38), the ␤-chemokine-containing T cells, and interferons (15). However, under the conditions CD40L-stimulated MDM supernatants were unable to protect tested, CD40L stimulated macrophages were not themselves MDM from infection by a macrophage-tropic NSI strain, protected from HIV infection nor did their supernatants ϩ HIV-1BaL, under conditions similar to those reported previ- protect CD4 T cells from infection by an SI strain of HIV-1. ously (15). Thus, the expression of CD40L alone might not completely CD40L Stimulation of MCP-1 Production by Macrophages. protect the host from all modes of HIV-1 replication. Because it was not feasible to examine all of the numerous Previously, only CD8ϩ T cells (33, 38) and CD4ϩ T cells (44, 45) have been considered as important immunologic sources of the HIV-1-suppressive ␤-chemokines. However, CD40L- stimulated MDM produced 5–20 times more MIP-1␣ and MIP-1␤ and only slightly less RANTES than any untrans- formed activated T cell yet described (33, 44, 45), when production is considered on a per cell per day basis. More importantly, Tedla et al. (46) used in situ hybridization and immunohistochemistry to show that the lymph nodes of HIV- 1-infected individuals contain abundant macrophages (most of which were not infected by HIV-1), which are producing MIP-1␣ and MIP-1␤. They also demonstrated RANTES expression by macrophages (although at a lower level than MIP-1␣ and MIP-1␤, similar to our in vitro results in Fig. 1B), as well as in perivascular cells. Although large numbers of T cells (mainly CD8ϩ T cells) were also present in these lymph

ϩ FIG. 2. Inhibition of NSI HIV-1 replication in CD4 T cells by CD40L-stimulated macrophage supernatants. CD4ϩ T cells (depleted of CD8ϩ T cells) were stimulated by PHA and IL-2, infected with the NSI isolate, (HIV-1SF162), and cultured for 14 days in either R10 medium or a 1:20 dilution of MDM supernatants. Virus production was measured by ELISA for HIV-1 p24 antigen. To demonstrate that the ␤-chemokines in the CD40L-stimulated MDM supernatant ac- counted for the HIV-1 suppressive activity, the supernatant was pretreated with a mixture of neutralizing antibodies against MIP-1␣, FIG. 3. CD40L stimulation of MCP-1 production by MDM. Cul- MIP-1␤, and RANTES, which abrogated its effectiveness. ture conditions were identical to that in Fig. 1B. Downloaded by guest on October 1, 2021 Immunology: Kornbluth et al. Proc. Natl. Acad. Sci. USA 95 (1998) 5209

FIG. 4. CD40L-macrophage pathway for the generation of ␤-chemokines. Stimulation of the T cell receptor (TCR) by peptidic antigen presented by major histo- compatibility complex (MHC) molecules (Signal 1) is sufficient to stimulate a subset of T cells to rapidly express CD40L (42). Macrophages bearing CD40 re- spond to cell–cell contact with such T cells by producing ␤-chemokines within 3 hr. The resulting ␤-chemokines may then act to attract additional lymphocytes and monocyte͞macrophages to the site of the developing immune reaction or block the infection of nearby CD4ϩ T cells by NSI strains of HIV-1.

nodes, they were not significant producers of any of these three in the extrinsic allergic encephalitis animal model (55, 56), T ␤-chemokines in these tissues (46). Therefore, macrophages cells expressing CD40L have been identified in direct contact should be considered as a major source of the HIV-1- with CD40-expressing macrophages in human brain lesions suppressive ␤-chemokines in vivo. In this context, our data (55) and MIP-1␣ has been identified in extrinsic allergic show that the level of macrophage ␤-chemokine production encephalitis lesions (57). CD40L has also been found in ϩ can be high enough to protect CD4 T cells against many of atherosclerotic plaques (58), where MCP-1 production by the NSI strains of HIV-1 that predominate during the early macrophages has also been demonstrated (39). Further indi- phase of HIV-1 infection (47), and we have identified CD40L rect evidence suggesting a linkage between CD40L expression as a stimulus for macrophage ␤-chemokine production. and ␤-chemokine production comes from animal studies Although we studied MCP-1 production because of its where anti-CD40L antibodies have been used to block cardiac, importance for immunologic lesions involving macrophages, skin, and renal transplant rejection (59) and chemically in- two recent studies suggest that there may be a role for MCP-1 duced immune colitis (60). In each case, not only was the in anti-HIV-1 immunity. An epidemiological study of the deleterious immunologic reaction prevented, but the infiltra- MCP-1 receptor, CCR2, revealed a delayed progression to tion of immune cells was markedly reduced as well. CD40L has AIDS in HIV-1-infected individuals who were homozygous for already been shown to be critical for the elaboration of a a mutation in the first transmembrane region (CCR2–64I) cellular immune response (23–27). In addition, our data now ͞ (48). Another study found that MCP-1 at 1 nM (8,700 pg ml) suggest that CD40L may act at the earliest phases of immune was found to suppress the replication of both NSI and SI strains reactivity to induce the ␤-chemokine-mediated recruitment of of HIV-1 in primary CD4ϩ T cell cultures (49). Although our ␣ additional cells into developing immunologic reactions or antibody neutralization studies demonstrated that MIP-1 , lesions. MIP-1␤, and RANTES could account for almost all of the HIV-1 suppressive activity in the supernatants of CD40L- We thank Sharon Wilcox, Darica Smith, Theresa Jackson, and Carol stimulated MDM (Fig. 2), our experiments tested relatively Latham for administrative support, and Violetta Alvarado, Sara high dilutions (1:20), which may have minimized any contri- Albanil, and Jeanne Aufderheide for p24 ELISA results. HIV-1SF162, bution from MCP-1. Similarly, the dilutions tested may have from Drs. Cecilia Cheng-Mayer and Jay A. Levy, was obtained from minimized any protective effects from the newly described the AIDS Research and Reference Reagent Program, Division of HIV-suppressive ␤-chemokine, macrophage-derived chemo- AIDS, National Institute of Allergy and Infectious Diseases, National kine (50). Institutes of Health (NIH). This work was supported by NIH Grants CD8ϩ T cell clones that express CD40L have been isolated RO1 AI35258 and RO1 HL57911 to R.S.K.; Grant Supplement P30 with high frequency from the peripheral blood of a subset of CA23100–15S1 to the University of California, San Diego (UCSD) HIV-1-infected individuals (51), and it is possible that such Cancer Center; and the State of California University-wide AIDS Research Program. Other support was provided by the UCSD Center cells contribute to the control of HIV-1 replication in early for AIDS Research (NIH Grant P30 AI36214) and the Research infection. Additional studies are needed to determine whether Center on AIDS and HIV Infection of the San Diego Veterans Affairs current anti-HIV-1 vaccines generate CD40L-expressing T Medical Center. cells in uninfected individuals or whether special immuniza- tion procedures are needed for their expansion. However, 1. Taub, D. D. & Oppenheim, J. J. (1994) Therapeutic Immunol. 1, because the control of HIV-1 replication by the immune 229–246. system is typically incomplete even in early infection, it is 2. Schall, T. J. & Bacon, K. B. (1994) Curr. Opin. Immunol. 6, relevant to note that HIV-1 infection and purified gp120 865–873. envelope protein have been reported to inhibit the expression 3. Murphy, P. M. (1994) Annu. Rev. Immunol. 12, 593–633. of CD40L on CD4ϩ T cells in vitro (52, 53), and that HIV 4. Sherry, B., Horii, Y., Manogue, K. R., Widmer, U. & Cerami, A. infection is associated with a preferential loss of CD40L- (1992) Cytokines 4, 117–130. expressing CD4ϩ T cells in vivo (54). This suggests that HIV-1 5. Schall, T. J., Jongstra, J., Dyer, B. J., Jorgensen, J., Clayberger, C., Davis, M. M. & Krensky, A. M. (1988) J. Immunol. 141, may have evolved a means to counter the CD40L-macrophage 1018–1025. pathway for ␤-chemokine production as a strategy to avoid the ␤ 6. Altman, L. C., Snyderman, R., Oppenheim, J. J. & Mergenhagen, suppressive effects of these -chemokines on viral replication. S. E. (1973) J. Immunol. 110, 801–810. These results are also important for immune diseases where 7. Verani, A., Scarlatti, G., Comar, M., Tresoldi, E., Polo, S., CD40L͞CD40 interactions may be involved. In multiple scle- Giacca, M., Lusso, P., Siccardi, A. G. & Vercelli, D. (1997) J. Exp. rosis, a disease in which CD40L is required for disease activity Med. 185, 805–816. Downloaded by guest on October 1, 2021 5210 Immunology: Kornbluth et al. Proc. Natl. Acad. Sci. USA 95 (1998)

8. Denis, M. & Ghadirian, E. (1994) Clin. Exp. Immunol. 96, Moore, J. P. & Paxton, W. A. (1996) Nature (London) 381, 187–192. 667–673. 9. Schmidtmayerova, H., Nottet, H. S., Nuovo, G., Raabe, T., 38. Moriuchi, M., Moriuchi, H., Combadiere, C., Murphy, P. M. & Flanagan, C. R., Dubrovsky, L., Gendelman, H. E., Cerami, A., Fauci, A. S. (1996) Proc. Natl. Acad. Sci. USA 93, 15341–15345. Bukrinsky, M. & Sherry, B. (1996) Proc. Natl. Acad. Sci. USA 93, 39. Yla-Herttuala, S., Lipton, B. A., Rosenfeld, M. E., Sarkioja, T., 700–704. Yoshimura, T., Leonard, E. J., Witztum, J. L. & Steinberg, D. 10. Canque, B., Rosenzwajg, M., Gey, A., Tartour, E., Fridman, (1991) Proc. Natl. Acad. Sci. USA 88, 5252–5256. W. H. & Gluckman, J. C. (1996) Blood 87, 2011–2019. 40. Chensue, S. W., Warmington, K. S., Ruth, J. H., Sanghi, P. S., 11. Lukacs, N. W., Kunkel, S. L., Strieter, R. M., Warmington, K. & Lincoln, P. & Kunkel, S. L. (1996) J. Immunol. 157, 4602–4608. Chensue, S. W. (1993) J. Exp. Med. 177, 1551–1559. 41. Pinchuk, L. M., Polacino, P. S., Agy, M. B., Klaus, S. J. & Clark, 12. Devergne, O., Marfaing-Koka, A., Schall, T. J., Leger-Ravet, E. A. (1994) Immunity 1, 317–325. M. B., Sadick, M., Peuchmaur, M., Crevon, M. C., Kim, K. J., 42. Casamayor-Palleja, M., Khan, M. & MacLennan, I. C. (1995) J. Galanaud, P. & Emilie, D. (1994) J. Exp. Med. 179, 1689–1694. Exp. Med. 181, 1293–1301. 13. Denis, M. (1995) Am. J. Respir. Crit. Care Med. 151, 164–169. 43. Caux, C., Massacrier, C., Vanbervliet, B., Dubois, B., Van 14. Petrek, M., Pantelidis, P., Southcott, A. M., Lympany, P., Sa- Kooten, C., Durand, I. & Banchereau, J. (1994) J. Exp. Med. 180, franek, P., Black, C. M., Kolek, V., Weigl, E. & du Bois, R. M. 1263–1272. (1997) Eur. Respir. J. 10, 1207–1216. 44. Conlon, K., Lloyd, A., Chattopadhyay, U., Lukacs, N., Kunkel, S., 15. Kornbluth, R. S., Oh, P. S., Munis, J. R., Cleveland, P. H. & Schall, T., Taub, D., Morimoto, C., Osborne, J., Oppenheim, J., Richman, D. D. (1989) J. Exp. Med. 169, 1137–1151. Young, H., Kelvin, D. & Ortaldo, J. (1995) Eur. J. Immunol. 25, 16. Bajorath, J., Seyama, K., Nonoyama, S., Ochs, H. D. & Aruffo, 751–756. A. (1996) Protein Sci. 5, 531–534. 45. Kinter, A. L., Ostrowski, M., Goletti, D., Oliva, A., Weissman, D., 17. Ho, S. D., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Gantt, K., Hardy, E., Jackson, R., Ehler, L. & Fauci, A. S. (1996) (1989) Gene 77, 51–59. Proc. Natl. Acad. Sci. USA 93, 14076–14081. 18. Noelle, R. J., Daum, J., Bartlett, W. C., McCann, J. & Shepherd, 46. Tedla, N., Palladinetti, P., Kelly, M., Kumar, R. K., DiGirolamo, D. J. (1991) J. Immunol. 146, 1118–1124. N., Chattophadhay, U., Cooke, B., Truskett, P., Dwyer, J., 19. Suttles, J., Evans, M., Miller, R. W., Poe, J. C., Stout, R. D. & Wakefield, D. & Lloyd, A. (1996) Am. J. Pathol. 148, 1367–1373. Wahl, L. M. (1996) J. Leukocyte Biol. 60, 651–657. 47. Connor, R. I., Sheridan, K. E., Ceradini, D., Choe, S. & Landau, J. Exp. Med. 185, 20. Cheng-Mayer, C. & Levy, J. A. (1988) Ann. Neurol. 23, S58–S61. N. R. (1997) 621–628. 48. Smith, M. W., Dean, M., Carrington, M., Winkler, C., Huttler, 21. Paxton, W. A., Martin, S. R., Tse, D., O’Brien, T. R., Skurnick, G. A., Lomb, D. A., Goedert, J. J., O’Brien, T. R., Jacobson, L. P., J., VanDevanter, N. L., Padian, N., Braun, J. F., Kotler, D. P., et al. (1997) Science 277, 959–965. Wolinsky, S. M. & Koup, R. A. (1996) Nat. Med. 2, 412–417. 49. Frade, J. M. R., Llorante, M., Mellado, M., Alcami, J., Gutierrez- 22. Hamilton, J. A. (1993) Immunol. Today 14, 18–24. Ramos, J. C., Zaballos, A., del Real, G. & Martinez-A., C. (1997) 23. Kroczek, R. A., Graf, D., Brugnoni, D., Giliani, S., Korthuer, U., J. Clin. Invest. 100, 497–502. Ugazio, A., Senger, G., Mages, H. W., Villa, A. & Notarangelo, 50. Pal, R., Garzino-Demo, A., Markham, P. D., Burns, J., Brown, 138, L. D. (1994) Immunol. Rev. 39–59. M., Gallo, R. C. & DeVico, A. L. (1997) Science 278, 695–698. 24. Ramesh, N., Morio, T., Fuleihan, R., Worm, M., Horner, A., 51. Paganelli, R., Scala, E., Ansotegui, I. J., Ausiello, C. M., Halapi, Tsitsikov, E., Castigli, E. & Geha, R. S. (1995) Clin. Immunol. E., Fanales-Belasio, E., D’Offizi, G., Mezzaroma, I., Pandolfi, F., Immunopathol. 76, S208–S213. Fiorilli, M., Cassone, A. & Aiuti, F. (1995) J. Exp. Med. 181, 25. Foy, T. M., Aruffo, A., Bajorath, J., Buhlmann, J. E. & Noelle, 423–428. R. J. (1996) Annu. Rev. Immunol. 14, 591–617. 52. Macchia, D., Almerigogna, F., Parronchi, P., Ravina, A., Maggi, 26. Van Kooten, C. & Banchereau, J. (1996) Adv. Immunol. 61, 1–77. E. & Romagnani, S. (1993) Nature (London) 363, 464–466. 27. Grewal, I. S., Borrow, P., Pamer, E. G., Oldstone, M. B. A. & 53. Chirmule, N., McCloskey, T. W., Hu, R., Kalyanaraman, V. S. & Flavell, R. A. (1997) Curr. Opin. Immunol. 9, 491–497. Pahwa, S. (1995) J. Immunol. 155, 917–924. 28. Alderson, M., Armitage, R. J., Tough, T. W., Strockbine, L., 54. Wolthers, K. C., Otto, S. A., Lens, S. M., Van Lier, R. A., Fanslow, W. C. & Spriggs, M. K. (1993) J. Exp. Med. 178, Miedema, F. & Meyaard, L. (1997) AIDS Res. Human Retrovi- 669–674. ruses 13, 1023–1029. 29. Kiener, P. A., Moran-Davis, P., Rankin, B. M., Wahl, A. F., 55. Gerritse, K., Laman, J. D., Noelle, R. J., Aruffo, A., Ledbetter, Aruffo, A. & Hollenbaugh, D. (1995) J. Immunol. 155, 4917– J. A., Boersma, W. J. & Claassen, E. (1996) Proc. Natl. Acad. Sci. 4925. USA 93, 2499–2504. 30. Stout, R. D., Suttles, J., Xu, J., Grewal, I. S. & Flavell, R. A. 56. Grewal, I. S., Foellmer, H. G., Grewal, K. D., Xu, J., Hardard- (1996) J. Immunol. 156, 8–11. ottir, F., Baron, J. L., Janeway, C. A., Jr., & Flavell, R. A. (1996) 31. Stout, R. D. & Suttles, J. (1996) Immunol. Today 17, 487–492. Science 273, 1864–1867. 32. Wagner, D. H., Jr., Stout, R. D. & Suttles, J. (1994) Eur. 57. Glabinski, A. R., Tani, M., Strieter, R. M., Tuohy, V. K. & J. Immunol. 24, 3148–3154. Ransohoff, R. M. (1997) Am. J. Pathol. 150, 617–630. 33. Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, 58. Mach, F., Schonbeck, U., Sukhova, G. K., Bourcier, T., Bonnefoy, R. C. & Lusso, P. (1995) Science 270, 1811–1815. J. Y., Pober, J. S. & Libby, P. (1997) Proc. Natl. Acad. Sci. USA 34. D’Souza, M. P. & Harden, V. A. (1996) Nat. Med. 2, 1293–1300. 94, 1931–1936. 35. Moore, J. P., Trkola, A. & Dragic, T. (1997) Curr. Opin. Immunol. 59. Larsen, C. P., Elwood, E. T., Alexander, D. Z., Ritchie, S. C., 9, 551–562. Hendrix, R., Tucker-Burden, C., Cho, H. R., Aruffo, A., Hol- 36. Broder, C. C. & Collman, R. G. (1997) J. Leukocyte Biol. 62, lenbaugh, D., Linsley, P. S., Winn, K. J. & Pearson, T. C. (1996) 20–29. Nature (London) 381, 434–438. 37. Dragic, T., Litwin, V., Allaway, G. P., Martin, S. R., Huang, Y., 60. Stuber, E., Strober, W. & Neurath, M. (1996) J. Exp. Med. 183, Nagashima, K. A., Cayanan, C., Maddon, P. J., Koup, R. A., 693–698. Downloaded by guest on October 1, 2021