Cellular interactions in lymphocyte proliferation: effect of syngeneic and xenogeneic macrophages.

Secondary cell-mediated responses to ectromelia virus infection were studied using an in vitro system. Lymphoid “responder” cells from mice which had recovered from intravenous primary infection at various times prior to sacrifice, were cultured with syngeneic, virus-infected macrophages or spleen cells as “stimulator” cells at 39 °C, a temperature which prevented the virus from exerting cytopathic effects against responder cells. This restrictive temperature and medium with 2-mercaptoethanol at 10^?4M often gave viable cell yields of more than 100% of the original responder cells over 4 days of culture. Preliminary experiments showed that spleen cells from primed mice, cultured with syngeneic, infected spleen cells from normal mice gave the most powerful secondary cytotoxic cell responses as measured by ⁵¹Cr release from virusinfected H-2-compatible target cells. The cytotoxic cells were sensitive to anti-θ and complement treatment and lysed H-2-compatible, virus-infected target cells much more efficiently than infected, allogeneic target cells, thus indicating that they were T cells. Some activity against uninfected H-2-compatible target cells was also generated, but this was largely independent of the presence of virus-induced antigen, (i.e. infected stimulator cells were unnecessary) and therefore seemed to be a consequence of the cultural conditions. Cold target competition showed that this activity was the responsibility of a T cell subset separate from the virus-specific cytotoxic T cells. The peak of cytotoxic activity against virus-infected targets occurred at 4 days of culture and DNA synthesis was maximal on day 3. The concentration of cytotoxic T cells at the peak was eight-fold higher than at the peak of the splenic primary response in vivo, Memory T cells (precursors of secondary cytotoxic T cells) appeared in spleen within 12–14 days of primary infection in vivo, reached a plateau at 5–6 weeks and persisted for at least 16 months. Spleen cells appeared partly refractory to secondary stimulation in vitro at 8–10 days post-priming. This did not seem to be due to cellular migration from spleen to lymph nodes or peritoneal cavity, but its cause was not determined. Primary responses in vitro were not detectable under conditions optimal for secondary responses, thus suggesting a major quantitative, or qualitative difference between virgin and memory T cells.     

Prostaglandin E2 production is enhanced in mice genetically selected to produce high affinity antibody responses

Spleen cells from mice selectively bred to produce high affinity antibody responses to protein antigens (HI) had reduced responses to both T and B cell mitogens when compared to those from mice selectively bred to produce low affinity (LO) responses. The reduced response by spleen cells from HI mice was partially reversed by the addition of indomethacin in vitro. Spleen adherent cells from HI mice had increased production of prostaglandin E2 when compared to those from LO mice. In addition, spleen adherent cells from mice which fail to show affinity maturation not only produced lower amounts of PGE2 than those from HI mice but also a decreased proportion of spleen cells adhered to plastic in these mice. To test the possibility that the increased PGE2 production in HI mice was responsible for the production of high affinity antibodies, indomethacin was administered in vivo and resulted in a significant reduction in antibody affinity. The possibility that PGE2 production may control the balance between the TH1 and TH2 cells of Mosmann and Coffman is discussed.     

The cell-mediated immune response to ectromelia virus infection. Secondary response in vitro: specificity, nature of effector and responder cells and requirements for induction of antigenic changes in stimulator cells.

An in vitro culture method was used to study secondary cell-mediated responses to ectromelia virus infection in mice. Infected, syngeneic spleen cells or peritoneal cells were efficient "stimulator" cells when cultured with "responder" cells obtained from mice infected with ectromelia 4-6 weeks previously. The kinetics of generation of cytotoxic cells in cultures were determined; a peak occurred on days 4-5. A separation procedure performed on the cytotoxic cells showed that activity was associated mainly with the Ig-negative subpopulation (T cell-rich) and that H-2 compatibility between cytotoxic cells and target cells was required. The secondary response was virus-specific, at the level of both induction and target cell lysis, at least so far as ectromelia and lymphocytic choriomeningitis (LCM) viruses are concerned. Seperation of responder cells prior to culture showed that a potent secondary response was generated with the Ig-negative (T cell-rich) subpopulation and only a weak response was observed when the responder cells were Ig-positive (rich in B cells). Infected stimulator cells did not appear to secrete significant amounts of soluble antigen into the medium over 4 days of culture. Thus, antigenic patterns effective in memory T cell stimulation may be largely associated with the surfaces of infected cells.Pretreatment of ectromelia virus with UV- or gamma-irradiation did not impair its ability to induce antigenic changes in stimulator cells. Stimulator cells treated with UV-or gamma-irradiated virus for 1 h and then immediately with pactamycin to inhibit further viral protein synthesis and replication were efficient stimulators, thus indicating that antigenic changes are induced very rapidly on the surface of stimulator cells after uptake of virus. These treatments are being used to further characterize the cellular requirements in the stimulator population.     

Resistance to fatal central nervous system disease by mouse hepatitis virus, strain JHM. II. Adherent cell-mediated protection

Resistance of SJL/J mice to intracranial inoculation with the JHM strain of mouse hepatitis, a coronavirus, is dependent upon the age of the animals at inoculation. Animals 12 weeks of age or older are resistant, whereas those 6 weeks or younger are uniformly susceptible to viral infection. Spleen cells or thioglycolate elicited peritoneal exudate cells can transfer resistance from 12-week-old to 6-week-old recipients. Removal of the adherent cells from either spleen or peritoneal cells ablated protection. Adherent cells from 12-week-old mice were protective even after depletion of Ia- and Thy-1-bearing cells. Antiviral antibody, thioglycolate injection into 6-week-old animals, and nylon wool-purified T cells were ineffective in mediating resistance. Adherent cells transferred 4 days before virus challenge, but not after challenge, were protective. Thus, there is an age-related change in SJL mice that protects from acute central nervous system disease, which may be due to maturation of a specialized adherent cell population.     

Suppression of parasite antigen-specific lymphoid blastogenesis in African trypanosomiasis

Inbred C57BL/6J mice were infected with either Trypanosoma rhodesiense organisms of Walter Reed Army Trypanozoon antigenic type 3 or 5 (WRATat 3 or WRATat 5) or were immunized with soluble trypanosomal antigens. Spleen cells obtained from immunized hosts undergo blastogenesis, measured by thymidine incorporation, when exposed to trypanosomal antigens in vitro. Spleens obtained from mice infected with T. rhodesiense organisms do not respond or respond only minimally to trypanosomal antigens in vitro. Spleen cells of infected mice suppress the trypanosomal antigen-induced proliferative response of spleen cells from immunized mice in co-culture experiments. The suppressive activity was found in both the plastic adherent and plastic nonadherent spleen cell populations. The in vitro responses of normal spleen cells to LPS and Con A were also suppressed by spleen cells obtained from infected mice.     

Requirements for stimulation of T cell responses against virus-infected cells: nature of ectromelia virus-infected cells capable of stimulating cytotoxic T cells in a secondary response in vitro.

The nature of infected stimulator cells in the in vitro secondary cytotoxic T cell response to ectromelia infection was investigated. It was found that macrophages were better stimulator cells than spleen cells. B cells (Ig-positive cells) were superior to T cells (Ig-negative cells) both on a relative proportion and on a cell-to-cell basis. Concanavalin A and lipopolysaccharide-stimulated lymphocytes were also effective stimulator cells but appeared to be slightly inferior to spleen cells. Spleen cells depleted of Ia-positive cells were markedly inferior to normal spleen cells as stimulators. It was also found that primary and secondary cytotoxic T cells were largely Ia-negative. These findings are discussed in relation to the likely events during T cell responses to infection in vivo.     

Neonatal susceptibility to MHV3 infection in mice. I. Transfer of resistance.

Up to 3 weeks of age, mice of the resistant A/J strain are fully susceptible to mouse hepatitis virus type 3 infection (MHV3). Immune deficiency, however, resulting from neonatal thymectomy or long term ALS administration led A/J animals to remain susceptible when tested at adult age. Whole spleen cells transferred from normal adult A/J donor mice protected suckling syngeneic recipients from i.p. infection with MHV3. Such a protective capacity of spleen cells was abolished after treatment with anti-theta serum and complement. Spleen cell separation by means of adherence to plastic also showed that neither the nonadherent nor the adherent populations injected separately were able to confer resistance to young mice challenged with the virus. Protection was not achieved with peritoneal cells originating from adult syngeneic animals. Transfer of resistance to MHV3 was obtained, however, when peritoneal cells were associated with adherent spleen cells. This study indicated that two types of mature cells, at least, were required for transferring MHV3 resistance into newborn mice of the A/J strain: T lymphocytes and an adherent spleen cell population.     

Selective impairment of B cell function by Neisseria meningitidis

Spleen cells from CBA/J mice infected with Neisseria meningitidis displayed depressed in vitro plaque-forming cell (PFC) responses to T-dependent (sheep red blood cell; SRBC) and T-independent (TNP-LPS, TNP-Ficoll) antigens. The inhibition was observed over a wide range of antigen concentrations. The decreased responsiveness of splenocytes from infected mice was due to a selective impairment of B-cell function since helper-T-cell activity was intact in infected mice as shown by the ability of T-enriched lymphocytes to cooperate with normal B-enriched lymphocytes in the generation of an anti-SRBC response, accessory macrophage function was preserved since adherent spleen cells from bacteria-injected mice were shown to produce normal or increased levels of IL-1 and were able to cooperate with normal non-adherent spleen cells in the generation of PFC against SRBC. Addition of peritoneal cells from normal animals or extraneous IL-1 both failed to restore normal PFC responses in cultures of splenocytes from infected mice. Finally, B-enriched lymphocytes from infected mice produced poor anti-SRBC responses when cultured with either Con A supernatant or T-enriched lymphocytes from normal or infected mice. Cell-mixing experiments failed to detect the presence of suppressor cells in cultures of unfractionated spleen cells or B-enriched lymphocytes from infected mice. Therefore, the immunological unresponsiveness associated with a Neisseria meningitidis infection was attributed to a meningococcus-induced defect(s) in B-cell function. In vivo polyclonal B-cell activation leading to clonal exhaustion did not play a major role in the depression of humoral responses since meningococcal infection induced little or no polyclonal Ig secretion.     

Suppression of responses to cryptococcal antigen in murine cryptococcosis

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.     

Sex-associated differences in the regulation of immune responses controlled by the MHC of the mouse

The immunologic potential of T lymphocytes and antigen-presenting cells (APC) from male and female mice were compared. Lymphocytes from female mice or from male mice that cannot produce and respond to testosterone (Tfm/y) were more reactive than male lymphocytes to alloantigens in MLR. Spleen cells from Tfm/y mice equipped with estrogen implants showed a higher responsiveness than control Tfm/y to alloantigens. The removal of suppressive adherent cells or the addition of T cell growth factor (TCGF) enhanced the proliferative activity of the cells in the MLR. The responsiveness of female cells to alloantigens, however, remained superior to that observed in male cells. Similarly, in the presence of TCGF, thymocytes from female mice react more effectively than male cells in MLR. In addition, Con A-stimulated spleen cells from female mice produce more interleukin 2 (IL 2) than do spleen cells from males or female mice treated with testosterone. Lymphocytes from immunized mice were tested for their ability to respond to soluble antigens (KLH and OVA) in vitro. Again, female immunocompetent cells respond more vigorously than male cells or cells originating in female mice with testosterone implants. APC from female spleen were more efficient than male APC in initiating a secondary response in primed lymphocytes from either males or female mice. Moreover, castration of male mice enhanced, and treatment of female mice with androgen reduced, the efficiency of antigen presentation. In conclusion, these data suggest that female cells are superior to male cells in immunologic functions that are known to be associated with reactions to and recognition of histocompatibility antigens, i.e., antigen presentation and MLR. Furthermore, our present data indicate that the differential reactivity of immunocytes between male and female mice depends on the hormonal balance of the animal.     

A role of HLA-DQ molecules of stimulator-adherent cells in the regulation of human autologous mixed lymphocyte reaction

In this study, we have found that treatment of stimulator autologous adherent cells with anti-HLA-DQ mAb resulted in markedly enhanced proliferative response of T cells in human autologous mixed lymphocyte reaction system wherein T cells were cultured with autologous adherent cells at near ratio of adherent cells to T cells in peripheral blood, in which T cells minimally proliferate. However, treatment of stimulator-adherent cells with anti-HLA class I, anti-DR and anti-DP mAb had no effect on the proliferative response of T cells under the condition. It was further observed that CD4-enriched cells could significantly proliferate in the presence of autologous adherent cells either untreated or treated with anti-DQ mAb, although treatment of adherent cells with anti-DR mAb blocked proliferative response of CD4-enriched cells. Moreover, the proliferative response of CD4-enriched cells was suppressed by addition of CD8-enriched cells in the presence of untreated adherent cells, whereas the proliferative response of CD4-enriched cells could not be suppressed by CD8-enriched cells when the adherent cells in the culture were treated with anti-DQ mAb. These observations suggest that CD8 T cells are required for suppression of proliferative response of CD4 T cells to HLA-DR molecules on autologous stimulator-adherent cells, and that HLA-DQ molecules on the surface of adherent cells play an important role in the induction of suppression by CD8 T cells.     

Bidirectional amplification of macrophage-lymphocyte interactions: enhanced lymphocyte activation factor production by activated adherent mouse peritoneal cells.

Adherent peritoneal cells (90 to 95% macrophages) from mice injected with Mycobacterium bovis, strain BCG, or certain noninfectious agents such as pyran copolymer or phytohemagglutinin showed increased chemotactic and tumoricidal capacity in vitro. These activated macrophages elaborated 2 to 5 times more lymphocyte-activating factor (LAF) in vitro than equal numbers of adherent cells from untreated mice. In contrast, adherent PC from mice treated with thioglycollate or mineral oil were not cytotoxic and did not produce more LAF than PC from untreated mice. Adherent PC from untreated nude mice, which have increased chemotactic and tumoricidal capacity in vitro, also exhibited enhanced LAF production compared to adherent PC from their normal littermates. Increased production of LAF was also evident with adherent PC and the macrophage-like tumor cell line P388D1 after incubation in vitro with bacterial endotoxins or with antigen-induced lymphokines. These data indicate that adherent PC can be activated either in vivo or in vitro to elaborate more LAF. Thus, activated macrophages are more effective than normal macrophages in amplification of the afferent limb of immune responses as well as in their effector functions.     

Virus-specific proliferative T-cell responses: Parameters and specificity

The genetic requirements for inducing virus-specific T-cell proliferation were investigated by taking spleen cells from animals primed with vaccinia virus in vivo, then culturing the cells in vitro with vaccinia virus-infected syngeneic peritoneal macrophages, and finally restimulating these cells a second time in vitro with vaccinia virus-infected macrophages from several strains of mice. Under these conditions, T cells proliferated in the tertiary response to virus-specific stimulation, whereas background proliferation caused by allogeneic differences between stimulator and responder cells was minimal. Compatibility between T cells and infected stimulator cells at the K or I regions alone or at I-A or I-A + I-B regions of the major histocompatibility complex (MHC) produced strong proliferative responses, whereas compatibility at D alone often resulted in somewhat weaker responses. However, these responses were rarely as great as in combinations of completely syngeneic stimulator and responder cells. Homology between responding and virus-infected stimulating cells in more than one of the H-2K, D, or I regions resulted in an additive, but not potentiating, effect. Genes coded outside the H-2 region did not seem to play a role in this system. In some rare cases, a weak response occurred across allogeneic barriers, but in general, virus-specific T-cell proliferation was strongly H-2 restricted.     

Requirement for non T-cells in the generation of cytotoxic T lymphocytes (CTL) in vitro. II. Characterization of the active cells in the spleen of nude mice.

Small numbers of LN cells will produce many more cytotoxic lymphocytes on in vitro culture with allogeneic stimulator cells if spleen cells from nu/nu mice are also present throughout the culture period. All cytotoxic cells produced are T cells and arise from precursors in the LN component. The nude spleen component appears to be providing a required non-T cell which has been lost from the LN component through dilution. At least two active subpopulations of cells, differing in sedimentation velocity, adherence properties, radiation sensitivity, and antigen recognition properties can be identified in the nu/nu spleen. The first, the dominant activity in normal nu/nu spleen, is nonadherent, radiation sensitive, and can synergize with either syngeneic or allogeneic LN cells provided both are different from the same alloantigens in the stimulator population. The second, found in nu/nu spleen cells precultured with alloantigen, sediments more rapidly, is adherent, and radiation resistant, and need not be allogeneic to the stimulator cells to synergize with LN cells. The first subpopulation may give rise to the second.     

BCG-induced suppressor cells. I. Demonstration of a macrophage-like suppressor cell that inhibits cytotoxic T cell generation in vitro.

Spleen cells obtained from mice 5 to 40 days after infection with viable BCG organisms (BCG-spleens) were found to be unresponsive in vitro to both mitogenic and alloantigenic stimuli. Moreover, suppressor cells could be demonstrated in the spleens from these infected animals. When spleen cells from BCG-infected mice were added to either syngeneic or allogeneic normal spleen cells, the mixtures neither proliferated nor developed cytotoxic activity when cultured with alloantigen or with concanavalin A (Con A). The development of unresponsiveness post-infection paralleled the onset of suppressive activity. Spleen cells obtained from mice given heat-killed BCG were neither suppressive nor unresponsive. The suppressive activity of BCG-spleen cells was associated with an adherent, phagocytic cell that lacked membrane-associated Thy-1 antigen. Removal of this cell by passage through nylon wool columns resulted in a cell population that was no longer capable of suppression and that responded normally to alloantigen and to Con A. It would thus appear that BCG infection results in the development of a "suppressor" macrophage-like cell population within the spleen. The role of this cell type in regulation of the immune response in BCG-infected animals is as yet undefined.     

Analysis of the cellular interactions involved in the regulation of induced erythrocyte autoantibodies

When normal mice are immunised with rat red blood cells (RBC) they produce autoantibodies against their own red blood cells as well as antibodies against determinants on rat RBC non-cross-reacting with self-RBC. Spleen cells from mice primed with rat RBC specifically suppress the subsequent induction of autoantibody in normal mice. The response to non-cross-reacting determinants on rat RBC is not affected. The results presented here indicate that either T cells or a thymus have to be present in the recipient for suppression to be manifest. It may therefore be the case that suppression is in fact induced in the recipient by the primed T cells.     

Vasoactive intestinal peptide stimulates T lymphocytes to release IL-5 in murine schistosomiasis mansoni infection.

In murine schistosomiasis, granulomas form around ova deposited in the liver and intestines of infected mice. The granulomas have eosinophils that produce vasoactive intestinal peptide (VIP) and T cells that display VIP receptors. IL-5 is a lymphokine important for the development and maturation of eosinophils. It seemed plausible that VIP, released from eosinophils, may interact with lymphocyte VIP receptors and modulate IL-5 production as part of a feedback regulatory circuit. Thus, we determined whether granuloma T cells make IL-5 and whether VIP modulates IL-5 production. Isolated granuloma cells enriched for T lymphocytes spontaneously released IL-5. Culture of these cells in the presence of VIP increased IL-5 secretion. Spleen cells were also studied. Spleen cells from infected mice did not spontaneously release IL-5 or express IL-5 mRNA and VIP did not stimulate these resting spleen cells to produce this IL. However, these cells did express IL-5 mRNA and secreted IL-5 in response to Con A or soluble egg Ag. VIP could not appreciably modulate IL-5 release when cells were cultured with VIP and the Ag or mitogen. Spleen cells washed free of Con A ceased IL-5 secretion within 24 h. These preactivated splenic T cells resumed vigorous IL-5 secretion in response to either Con A or VIP. Yet only Con A prominently induced IL-5 mRNA expression. VIP was an effective stimulus at concentrations equal to or above the kDa of the VIP receptor on both splenic and granuloma T cells (10(-8) M). It is concluded that, in murine schistosomiasis, VIP invokes IL-5 release from activated T cells that are not undergoing immediate TCR stimulation.     

Induction of cytotoxic T lymphocytes against herpes simplex virus type i: role of accessory cells and amplifying factor

Spleen cells from mice primed with herpes simplex virus type 1 (HSV-1) could be induced to differentiate into cytotoxic T lymphocytes (CTL) by in vitro culture with infectious HSV-1 but not by heat-inactivated virus. Induction of CTL failed to occur if the spleen cells were depleted of adherent cells by passage over columns of nylon wool before culture with virus. The CTL response could be restored by adding normal syngeneic peritoneal cells (PC) or L cell fibroblasts but not by allogeneic PC or BALB/c 3T3 fibroblasts. Thus, the induction of HSV-1-specific CTL was H-2 restricted. The response of HSV-1-stimulated nylon wool-depleted spleen cells could also be restored by adding amplifying factor (AF) produced in 24 hr mixed lymphocyte cultures. The addition of AF to nondepleted spleen cells also permitted the generation of CTL with heat-inactivated HSV-1 as a viral stimulant. Our results indicated that induction of a HSV-1 CTL response requires two signals, one provided by virus and a second, presumably nonspecific, by helper T cells. It was suggested that only the helper cells require H-2 restriction and need to be presented virus in the context of a macrophage.     

In vitro regulation of the pathogenic autoantibody response of New Zealand black mice. I. Loss with age of suppressive activity in T cell populations

An investigation of the regulation of specific anti-self responses was initiated with the development of an in vitro system in which spleen cells from NZB mice were stimulated by syngeneic mouse erythrocytes (MRBC) to produce MRBC-specific autoantibody-secreting cells. The response was measured by a modification of the focus-forming cell (FFC) assay, which enumerates cells secreting IgG, which specifically bind MRBC. Spleen cells from 9- to 12-mo-old NZB mice developed MRBC-specific FFC after 3 to 5 days in culture with MRBC. Few FFC were detected in the absence of MRBC in culture. Spleen cells from young (1- to 4-mo-old) NZB mice developed few if any FFC. Spleen cell populations containing T cells from young NZB mice suppressed this anti-MRBC response, whereas B cell populations from these young mice did not. In contrast, spleen cells, including T cell-enriched populations from old, Coombs'-positive mice were not capable under the same conditions of producing equivalent suppression of this in vitro autoimmune response. These data suggest that a population of suppressor T cells that may control the autoimmune anti-MRBC response in young NZB mice is lost, or else its activity is masked in old NZB mice that are actively producing anti-MRBC antibody.     

Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.