Enhancement of IGE-induced mediator release from rat peritoneal mast cells by serum-treated zymosan

Phagocytosis of zymosan (Z) treated with rat serum (ZX) by rat peritoneal mast cells caused only a small amount of [3H]serotonin release, and prior release of mediators from mast cells did not affect phagocytosis of sheep erythrocytes bearing IgG and C3b, indicating the independence of these two phenomena. When, however, mast cells were exposed to ZX, subsequent IgE-mediated release of histamine, [3H]serotonin, and beta-hexosaminidase was greatly enhanced. Prevention of complement activation by the presence of EDTA during the treatment of Z with the serum or prior heating of the serum at 56 degrees C for 30 min only slightly impaired the ability of ZX to augment mediator release, whereas prior absorption of the serum with zymosan at 0 degree C greatly diminished the enhancement. Exposure of fresh Z to variable amounts of either the acid or the high-salt eluate of ZX also generated ZX capable of enhancing [3H]serotonin release in a dose-dependent fashion. IgG, IgM, C3, and albumin were detected in the eluates by immunodiffusion. When IgG was depleted from the high-salt eluate by treating with Sepharose-anti-IgG, the enhancement was significantly reduced, indicating that IgG but not C3 or other immunoglobulins was required for the enhancement.     

Different roles of IgG and complement receptors in phagocytosis by polymorphonuclear leukocytes.

The functions of IgG and complement receptors in phagocytosis of immune complexes by mouse polymorphonuclear leukocytes were examined by in vitro experiments. The immune complexes were sheep red cells (E) sensitized with IgG antibody (EA) or with antibody and complement (EAC). Inhibition experiments with Fab fragments of rabbit IgG antibody anti-mouse IgG have shown that the complement receptor is primarily involved in the attachment phase, whereas participation of the IgG receptor is necessary for inducing the mechanism of phagocytosis. The possible relevance of these findings for the in vivo mechanism of defense infection, and for the control of antibody synthesis is discussed.     

Neutrophil receptors for IgG and complement: their roles in the attachment and ingestion phases of phagocytosis.

The functional roles of IgG and C3b in phagocytosis by human peripheral neutrophils were investigated. Phagocytosis of Staphylococcus aureus in the presence of human serum was severely depressed by heat inactivation of serum at 56 degrees C for 30 min. Experiments with varying particle: leukocyte ratios in the presence of complement-inactivated sera showed that particle-bound C3b can mediate a 10-fold enhancement of the overall phagocytic rate. When sheep erythrocytes were sensitized with either IgG or IgM, only the former were bound to and readily internalized by neutrophils. Erythrocytes sensitized with both IgM and C3b were bound but not internalized. Furthermore, the presence of Fc fragments during incubation of S. aureus or latex beads with neutrophils in the presence of IgG or fresh serum affected a total inhibition of internalization but did not significantly alter adherence. Quantitative data regarding IgG sensitization indicated that bound C3b results in at least a 3-fold decrease in the amount of sensitizing IgG required for 50% maximal phagocytic response by neutrophils. On the basis of the above results, it is argued that particle-bound C3b functions primarily in the adherence phase and that bound IgG serves as a trigger for the internalization phase of phagocytosis.     

Is the membrane attack complex of complement an enzyme?

Summary Recent studies on the functional activities of the membrane attack complex of complement, C5b-9, are reviewed. A new speculative hypothesis has been advanced to account for the ability of complement to mediate lysis of various targets. This hypothesis has three major elements: 1) that the membrane attack complex is an enzyme; 2) that the substrate for this putative enzyme is a membrane constituent; 3) that the substrate specificity of the putative enzyme is dependent on the species source of individual complement components within the C5b-9 complex.Abbreviations E = sheep red cells - A = rabbit IgM anti-Forssman antibody - Hu or hu = human - GP or gp = guinea pig     

Complement-mediated hemolytic activity of succinylated concanavalin A: preparation and activity of cell intermediates

Succinylated concanavalin A (SCon A) lyses sheep erythrocytes (E) in the presence of complement, whereas the native tetravalent lectin, Con A, is inactive. We have studied the ability of E-SCon A (ES) to interact with early acting guinea pig (gp) or human (hu) complement components (C1, C2, C4) and found that cell intermediates ESC1, ESC4, ESC14, and ESC142 can be generated that are analogous to intermediates conventionally prepared with E and rabbit IgM (pentameric) anti-Forssman antibody. Titration of gp or hu C1, C4, and C2, and quantification of the number of activated C1 molecules bound to ESC4 by the C1 fixation and transfer test showed in each case that an average of one effective site per cell was sufficient to cause cell lysis. Determination of tmax for optimal formation of ESC142 sites depended on the species combination of components used to make the intermediates, and the decay of ESC142 and EAC142 sites or sites generated with ESC4, EAC4, and trypsin-activated C2 were similar. The sugar alpha-D-methylglucopyranoside (alpha-MGP) inhibited binding of SCon A to E and eluted the lectin from ES, whereas galactose was nearly inactive, consistent with lectin sugar-binding selectivity. In contrast, both sugars were ineffective in eluting SCon A or C4hu from ESC4hu, indicating that C4hu blocked the interaction between lectin and alpha-MGP, perhaps by steric hindrance. SCon A is a divalent functional analogue of IgM anti-Forssman antibody that may be a uniquely suited reagent specific for cell membrane glycoconjugates for studying the mechanism of binding and activation of complement components.     

Phagocytosis by mouse peritoneal macrophages plated on monoclonal antibody-coated immune complex-substrates: effects of complexes of different IgG subclasses on Fc receptor functions

Macrophages plated on immune complex-coated substrates of different mouse IgG subclasses were examined for their capacities to phagocytose sheep red blood cells (SRBC) coated with monoclonal antibodies (MAb) of various IgG subclasses. IgG2a-and IgG2b-coated substrates abrogated macrophage phagocytosis of particles coated with any of the four mouse IgG subclasses. These results were confirmed by the use of two MAb of each of the IgG2a and IgG2b subclasses, with one of the MAb specific for dinitrophenyl groups and the others for SRBC. IgG3-coated substrates reduced the macrophage uptake of IgG2a-but not IgG2b-coated particles. Rabbit IgG-coated substrates ablated the uptake of SRBC coated with all mouse IgG subclasses. Resident and thioglycollate-elicited macrophages showed similar phagocytosis reduction when plated on these immune complexes. The phagocytosis of complement-coated particles was not affected by these IgG-coated substrates. Macrophages plated on both IgG2a-and IgG2b-coated substrates showed reduced immunofluorescence staining by an anti-IgG2b Fc receptor (FcR) Ab, 2.4G2 and reduced E(IgG2a) and E(IgG2b) binding. The results show that substrates coated with various IgG subclasses can abrogate phagocytosis mediated by FcR that do not have binding specificity for the substrate-immobilized Fc ligand, and suggest that the three classes of mouse FcR co-modulate.     

Extracellular release of rat eosinophil peroxidase (EPO) I. Role of anaphylactic immunoglobulins

The release of intracellular peroxidase (EPO) was investigated in order to evaluate rat eosinophil activation by various immunoglobulin (Ig) isotypes. After successive incubations with purified rat IgG1, IgG2a, IgG2b, IgG2c, IgE, or IgM and their respective anti-Ig antisera, eosinophils released significant amounts of EPO (up to 26% of the intracellular content) only in the case of Ig with anaphylactic activities (IgG2a and IgE). Other classes and subclasses were unable to induce EPO exocytosis. Selective depletion and reconstitution experiments suggested that mast cells were not required in this process. Similar levels of EPO could be released after interaction of eosinophils with antigen-antibody complexes (IgG2a monoclonal antibody and Schistosoma mansoni antigen) immobilized on nonphagocytosable surfaces. These results indicate that EPO exocytosis can be obtained after cell activation with specific antibodies, and that this mechanism is independent of phagocytosis. A kinetic study of eosinophils from S. mansoni-infected rats revealed that IgG2a and IgE cytophilic antibodies induced EPO release after incubation with either specific antisera or specific antigen, which suggests the in vivo relevance of such findings. The present work underlines the parallelism of interaction of anaphylactic-type Ig with eosinophils and with mast cells. Moreover, EPO release seems to represent an interesting marker of eosinophil activation, because close relationships were established between the present findings and previous work on the effector function of rat eosinophils.     

Suppression of B cell development and antibody responses in mice with polyclonal rabbit and monoclonal rat anti-IgM antibodies. I. Characterization of the suppressed state

Mice treated from birth with polyclonal, crude or affinity purified rabbit or monoclonal rat anti-mouse IgM antibodies [b-7-6 and C-2-23: Eur. J. Immunol. 14: 753-757, 1984] were found to be heavily suppressed with respect to B-cell activities. Crude or affinity purified rabbit or monoclonal rat anti-mouse IgM gave comparable results as follows: serum IgM was below detectable levels; serum IgG was reduced to about 1-3% of normal levels; free anti-IgM was always detectable; IgM and/or kappa-light-chain positive cells as well as IgM-secreting cells were absent in various lymphoid organs; the B-cell mitogen lipopolysaccharide was unable to induce proliferative responses; primary antibody responses could not be induced against sheep red blood cells and phosphorylcholine; lymphoid organs were reduced in size and B-cell areas were not populated with lymphocytes; besides a 40% reduction in absolute lymphocyte numbers in the blood, we found increased platelet counts and a 10% eosinophilia in anti-IgM-treated mice.     

Alternative complement pathway activation by C4b deposited during classical pathway activation

Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.     

Studies on the neutralization of herpes simplex virus. XI. Differences between slow-reacting complement-requiring neutralizing (s-CRN) antibodies in IgG and IgM

Late IgG and IgM from a rabbit immunized with herpes virus were tested for ordinary neutralizing (N) antibody, complement-requiring neutralizing (CRN) antibody and in addition CRN antibody detectable by overnight sensitization at 0 C (s-CRN antibody). Heat stability tests showed that IgG s-CRN antibody was slightly less resistant to heating at 70 C than were N and CRN antibodies, whereas all three activities of IgM were quickly degraded at this temperature. Dose-response curves with varying amounts of complement (C) or anti-antibody revealed a marked difference between IgG s-CRN and IgM s-CRN antibodies. While 1-hr sensitization at 37 C was insufficient to detect IgG s-CRN antibody, it had the same effect as overnight sensitization at 0 C for IgM s-CRN antibody. When sensitization at 0 C was prolonged to 3 days, unexpectedly high endpoints exceeding 1:10,000 were obtained even with IgM. consequently, enhancement by C was several hundred-fold with IgM in contrast to 5- to 10-fold enhancement of IgG s-CRN antibody, which was similar to that after overnight sensitization. Also IgM obviously required more C than did IgG. These results suggest that IgM of late serum is slower reacting and more C-dependent than IgG s-CRN antibody. Tests with early rabbit sera indicated that the s-CRN antibody detectable by 3-day sensitization reaches a high level before the appearance of N antibody.     

RNA conversion of lymphoid cells to synthesize allogeneic immunoglobulins in vivo

Ribonucleic acid extracts (“5 day immune” and “nonimmune”-RNA) obtained from lymph nodes and spleens of rabbits homozygous for the b⁴ or b⁵ allele of light chain immunoglobulin allotypes were injected iv into nonimmunized rabbits homozygous for the alternate allele. The recipient rabbits were then given multiple iv injections of sheep red blood cells (SRBC). The spleens were assayed 13, 21, and 37 days following the RNA injection for “direct” IgM and “indirect” IgG plaque forming cells (PFC) specific for SRBC. The b4 or b5 light chain allotype and the a1, a2, and a3 heavy chain allotype of the antibody in the plaques was identified by radioautography and by inhibition of plaque formation using anti-allotype antibodies. The b light chain allotype of the RNA donor was identified in 22–32% of the IgM plaques and in 25–42% of the IgG plaques. The allotype of the host rabbit b light chain allotype was identified in 56–67% of the IgM plaques and in 57–71% of the IgG plaques. Likewise the a heavy chain allotype of the RNA donor was identified in 10–19% of the IgM plaques and in 12–19% of the IgG plaques. The allotype of the host rabbit a heavy chain allotype was identified in 51–60% of the IgM plaques and in 55–63% of the IgG plaques. The concentrated lysates of spleen and lymph node cells were also analyzed for immunoglobulins of each light chain allotype by immunodiffusion with radiolabeled antibody. The allotype of both the RNA donor rabbit and host rabbit were found in most of the lysates of lymphoid tissues and in some of the IgG isolated from the serum and concentrated.     

Alterations in intramembrane particle distribution during interaction of erythrocyte-bound ligands with immunoprotein receptors

Freeze fracture studies have been performed on rabbit pulmonary alveolar macrophages and a nonphagocytic murine lymphoblastoid cell line, PU-5 Fc+, incubated with sheep erythrocytes, sheep erythrocyte-IgG Forssman antibody complex, sheep erythrocyte-IgG Forssman antibody-C complexes and aggregated IgG. Alveolar macrophages show redistribution of intramembrane particles after interaction with (EIgG) and E(IgM)C. The murine lymphoblastoid cell line shows intramembrane particle redistribution consequential to binding of E(IgG) and aggregated IgG. The results demonstrate that after specific immunoprotein receptor-ligand interaction, there is extensive plasma membrane reorganization which results in a redistribution and loss of intramembrane particles. Changes are observed in the protoplasmic face of the plasma membrane after the binding of ligand to the outer membrane surface. The findings suggest that interaction of erthrocyte-bound ligands with specific lymphoid and macrophage plasma membrane receptors leads to a generalized redistribution of integral membrane components in the membrane.     

IgG Suppresses Antibody Responses in Mice Lacking C1q,C3, Complement Receptors 1 and 2, or IgG Fc-Receptors

Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes.     

Triggering of histamine release from rat mast cells by divalent antibodies against IgE-receptors.

Antibodies against receptor molecules for IgE on rat basophilic leukemic (RBL) cells were prepared by immunization of a rabbit with immune precipitates composed of IgE-receptor complexes and anti-IgE. Antibodies against cell surface components were specifically purified by using RBL cells and rendered specific for mast cells by appropriate absorption. The major antibodies in the final preparation (anti-RBL) were directed against receptor molecules. It was found that the F(ab')2 fragments of anti-RBL induced histamine release from rat mast cells and caused immediate skin reactions in normal rats. These reactions by anti-RBL or its F(ab')2 fragments were inhibited if the receptors on mast cells had been saturated with IgE. The Fab' fragments of anti-RBL could bind with receptors on RBL cells and blocked passive sensitization of mast cells with IgE antibodies, but failed to induce skin reactions and histamine release from normal mast cells. Sensitization of normal rat skin with the Fab' fragment followed by an i.v. injection of anti-rabbit IgG induced skin reactions. The results indicated that bridging of receptor molecules by divalent anti-receptor antibody triggered mast cells for histamine release.     

Fibronectin-enhanced phagocytosis of an alternative pathway activator by human culture-derived macrophages is mediated by the C4b/C3b complement receptor (CR1)

We examined the ability of human monocytes and culture-derived macrophages under serum-free conditions to phagocytose desialated sheep erythrocytes (E), an activator of the alternative pathway of human complement. Freshly derived monocytes ingested desialated erythrocytes, but the degree of phagocytosis varied among individual donors. However, exposing the phagocyte to intact plasma fibronectin (Fn) had no effect on monocyte phagocytosis. Macrophages derived from monocytes in culture were far more efficient at ingesting desialated E, and the extent of phagocytosis was proportional to the degree of desialation. Although exposure of macrophages to substrate-bound Fn or fluid-phase Fn enhanced the phagocytosis of desialated E, pretreatment of desialated E with Fn did not enhance phagocytosis, demonstrating that Fn acted through an interaction with the macrophages. Fn-enhanced phagocytosis of desialated E was inhibited by treating macrophages with a monoclonal antibody to the C4b/C3b receptor (CR1), but not with a monoclonal antibody to the receptor for C3bi (CR3). Addition of cobra venom factor (CVF) to the macrophages also inhibited Fn-enhanced phagocytosis of desialated E. Phagocytosis of IgG-sensitized E, either in the absence or in the presence of Fn, was not significantly affected by anti-CR1 or CVF, demonstrating that these reagents did not lead to a general inhibition of phagocytosis. These experiments suggest that macrophages may deposit enough C3b onto desialated E to cause CR1-mediated phagocytosis in the presence of Fn. The ability of macrophages to opsonize and ingest foreign particles that activate complement may be critically important in areas of inflammation where concentrations of serum-derived specific opsonins may be inadequate.     

Receptors for IgM on certain human B lymphocytes.

A receptor for IgM was demonstrated on the surface of human B lymphocytes by using a rosette technique with ox erythrocytes coated with rabbit IgM antibody (EAM). Lymphocytes forming rosettes with EAM did not bind sheep red cells, had membrane Ia-like antigens and, in some instances, surface immunoglobulin. The specificity of EAM rosettes was confirmed by inhibition experiments with purified human Ig. IgM but not IgG molecules inhibited the rosette reaction. In addition, inhibition of EAM rosettes with IgM fragments showed that the receptor has affinity for a part of the molecule located in the Fc portion. By analogy with the receptors previously found on certain human T cells, receptors for IgM were not detected on freshly isolated B cells, but were expressed after overnight culture in IgM-free media. Studies on different human lymphoid tissues showed that IgM receptors are expressed on a limited percentage of both circulating and noncirculating B cells. In addition to normal B cells, the malignant B cells of a majority of cases of chronic lymphocytic leukemia expressed the receptors for IgM.     

Anaphylactic properties of mouse monoclonal IgG2a antibodies

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 $\text{K}\text{D}$ antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.     

Complement receptor type three-dependent degradation of opsonized erythrocytes by mouse macrophages

The role of the complement receptor type 3 (CR3) on thioglycollate-elicited peritoneal macrophages (TG-PM) in the destruction of opsonized particles was studied. We found that sheep red blood cells (E) that were opsonized with an IgM monoclonal anti-Forssman antibody and complement (E-IgM-C) were lysed by TG-PM, whereas there was little lysis of E pretreated with either the antibody or the complement source alone. Furthermore, this lysis could be inhibited by anti-CR3 monoclonal antibodies that had previously been shown to inhibit binding of E-IgM-C to the CR3. Kinetic studies of phagocytosis and lysis indicated that lysis of E-IgM-C occurs after phagocytosis, suggesting that lysis is an intracellular event. Further findings suggested that intra-cellular lysis was promoted by CR3 bound to the phagocytosed target, because a monoclonal anti-CR3 antibody decreased the rate of phagocytosis of E-IgM-C but not its magnitude, whereas the rate and extent of lysis were strikingly inhibited. Furthermore, TG-PM that had already internalized unopsonized E selectively lysed E-IgM-C that were added later. These data confirm that the interaction of the CR3 with its ligand on E-IgM-C promotes rapid phagocytosis, and further suggest that the CR3 facilitates degradation of the target particle once internalization has occurred.     

Protection against Campylobacter jejuni infection in suckling mice by anti-flagellar antibody

We obtained two monoclonal antibodies of IgM class and IgA class of immunoglobulin prepared from mouse spleen cells immunized with crude flagellar preparation, and a polyclonal antibody raised against purified flagellin monomer of Campylobacter jejuni in a rabbit. The specificity of the reaction of these antibodies for flagellar filament was confirmed by Western blotting and by immunoelectron microscopy. These antibodies caused agglutination of the bacteria and inhibited the motility of the bacteria. When a strain of C. jejuni was treated with IgM class monoclonal antibody before being inoculated into suckling mice, it reduced colonization of the intestinal tract by this bacteria. Inhibition of the colonization by IgA class monoclonal antibody was less effective than that of IgM class, and the polyclonal antibody consisting mostly of IgG class immunoglobulin was without effect.     

Immunoglobulins and complement in the skin of rhesus monkeys immunized with x-irradiated cercariae of Schistosoma japonicum

Skin sections of rhesus monkeys immunized with X-irradiated Schistosoma japonicum cercariae were stained by an unlabeled antibody enzyme method for the detection of IgG, IgM, IgA, IgE, and C3. While all of them could be found in the skin lesions, their distribution patterns varied with time and reactions. In whealing reaginic reactions, IgE appeared prominent, having an affinity to the mast cells. In Arthus-like reactions, IgG was predominant. IgG complexes were found on the schistosomula, on the walls of blood vessels, and in granulocytes near the schistosomula. In the late stage of Arthus-like reactions and in delayed hypersensitive reactions, IgA was predominant in granulocytes, mononuclear cells, and macrophages. Characteristics of each immunoglobulin pattern seemed to reflect its function in the effector mechanism. It may be speculated that these immunoglobulins and C3, together with effector cells, synergistically and sequentially destroy schistosomula in the skin.